Antisense oligonucleotides for treating allergy and neoplastic cell proliferation

ABSTRACT

Antisense oligonucleotides for treating and/or preventing at least one of asthma, allergy, hypereosinophilia, general inflammation and cancer are provided. The oligonucleotides are directed against nucleic acid sequences coding for a receptor selected from the group consisting of a CCR3 receptor and a common sub-unit of IL-3, IL-5 and GM-CSF receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Stage of International ApplicationNo. PCT/CA2005/001656 filed on Oct. 25, 2005, which designated Canada,and which claims the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalApplication No. 60/623,206 filed on Oct. 29, 2004.

FIELD OF THE INVENTION

The invention relates to the use of antisense oligonucleotides directedagainst specific cellular receptors, alone or in combination, in orderto inhibit general inflammation, including inflammation associated withasthma and allergy, and hypereosinophilia. The invention also relates tothe use of antisense oligonucleotides to inhibit neoplastic cellproliferation such as cancer.

BACKGROUND OF THE INVENTION

Antisense oligonucleotides are a new class of pharmaceuticals. Ingeneral, antisense refers to the use of small, syntheticoligonucleotides, with the same constituents as that found in human DNAor RNA. The antisense oligonucleotides are designed as a complementarysequence of a part of a gene they are targeting in order to be able toadhere to this sequence and inhibit gene expression. Gene expression isinhibited through hybridization of an antisense oligonucleotide to aspecific messenger RNA (mRNA) sense target according to the Watson-Crickbase pairing in which adenosine and thymidine (uracile in mRNA) orguanosine and cytidine interact through hydrogen bonding. Two mechanismscan account for these effects, the first being hybridization withimpaired translation of targeted mRNA, the second being the induction ofRNase H or similar enzymes with degradation of mRNA. A major advantageof this strategy is the specificity of action with the potential forless side effects and toxicity, especially when applied to the site ofaction (topical treatment). This therapeutic strategy could potentiallybe applied to any disease where an over-expression of one or severalgenes is believed to cause the presence or persistence of the disease.As a result, there have been numerous studies of antisenseoligonucleotides as therapeutic agents for cancer and viral diseases.

Antisense oligonucleotides can be used to inhibit interleukin (IL)-6receptor expression and thus the effects of the acute inflammatorymediator interleukin-6 on cells. Few studies have been conducted toassess whether antisense oligonucleotides can be employed to inhibitother receptors on cells that are involved in inflammation, including,but not limited to inflammation associated with asthma and inflammationassociated with atopic diseases and allergy or on cancerous cells.

Asthma is a disease that affects 5 to 10% of the population that hasdoubled in prevalence in the last 25 years. This increase has been notedespecially in infants after a viral infection of the airways(bronchiolitis), in children and in occupational induced asthma. Therecurrent breathing problems associated with asthma are often triggeredby allergens but the exact cause of asthma is not yet known. However, itis believed that agents such as viruses are involved in the perpetuationof the abnormal inflammation that is found in the airways of patientswith asthma and thus the persistence of the disease.

For this reason the current recommendations for first line therapy ofasthma is a potent anti-inflammatory medication such as those containingcorticosteroids and anti-leukotrienes. Although this therapy iseffective in many patients, some patients are resistant tocorticosteroids. This medication is also a potent immunosuppressive withlong term side effects and has not been shown to be effective in theprevention of allergy or asthma. Anti-leukotrienes have some effect inallergy and asthma but are not as effective as corticosteroids.

Several inflammatory mediators play a role in the appearance andperpetuation of inflammation in the airways of patients with asthma.Some mediators attract the inflammatory cells into the airways eitherthrough chemotaxis of eosinophils (the chemokines: RANTES, eotaxins 1,2, 3, MCP-3, 4 that act mostly in asthmatic inflammation through areceptor called CCR3) or through endothelial cell activation (IL-4,-13). Other mediators cause the priming and increased survival ofinflammatory cells in the airways (IL-3, -4, -5, GM-CSF). Thesemediators thus consist of either specific chemokines for eosinophils orof cytokines of the T helper lymphocyte type 2 phenotype (Th2: IL-3, -4,-5, -6, -9, -10, -13 and GM-CSF), (John A E. and Lukacs N W., 2003Sarcoidosis Vasc Diffuse Lung Dis., 20:180-189; Blease et al., 2003,Expert Opin Emerg Drugs. 8:71-81). An improvement, in asthma and generalrespiratory inflammation, has been shown when there is a decrease inthese inflammatory mediators in the airways.

Allergy is a hypersensitivity to an allergen causing an undesirableimmune response. Allergy is a disease that is extremely prevalent, forexample atopic rhinitis and conjunctivitis affect around 30% of thepopulation. Allergy is characterized by abnormal IgE production andinflammation to an allergen. In the presence of IgE and allergen,effector cells, such as the mast cells degranulate and releaseinflammatory mediators leading to the recruitment of the sameinflammatory cells that are found in asthma. In allergic rhinitis (i.e.hayfever), allergic conjunctivitis, nasal polyposis, chronic sinusitisand eczema, such as atopic dermatitis, one finds the same excess ininflammatory mediators as those present in asthma. IL-4 and IL-13 arenecessary for the production of IgE and the induction of the cells witha Th2 phenotype (Barnes P J., 2003, Cytokine Growth Factor Rev.14:511-522; Schuh et al., 2003, Cytokine Growth Factor Rev. 2003,14:503-510). Atopic diseases is a generic name for allergic diseaseswhich are developed by exposure to allergens, especially in individualswith a genetic propensity for being easily sensitized to allergens.Individuals having these predisposing factors easily develop an abnormalimmune response to alimentary antigens and inhalants. Some specificexamples of allergic diseases are bronchial asthma, a topic dermatitis,urticaria, allergic rhinitis, allergic conjunctivitis and allergicenterogastritis.

A neoplasm is an abnormal tissue growth that is uncontrollable andprogressive. A malignant neoplasm is often characterized as a cancer.Cancer is the second leading cause of death in humans and is a generalterm for more than 100 diseases characterized by abnormal proliferationof immortalized cells. One of the mechanisms that is involved in thepersistence and increase in these cells is by the release of growthfactors that act through receptors and lead to cellular proliferation.Amongst these growth factors, GM-CSF has been shown to be an importantgrowth factor for several tumour cells. The chemokine receptor CCR3 wasrecently characterized in malignant B lymphocytes recovered frompatients with chronic lymphocytic leukemia (CLL) and with hairy cellleukemia (HCL), (Trentin et al., 2004, Blood, 104, 502-508). Indeed, thetransactivation of Epidermal Growth Factor Receptor (EGFR) through CCR3chemokine receptor was found to be a critical pathway that elicits MAPkinase activation and cytokine production in bronchial epithelial cells(Adachi et al., 2004, Biochem. Biophys. Res. Commun. 320, 292-396). Theinhibition of proliferation of cancerous cells by blocking the receptorsfor growth factors and/or for chemokines, may be important in thetherapy of certain cancers.

Eosinophils are a type of white blood cell. They are granular leukocyteswith a nucleus that usually has two lobes connected by a slender threadof chromatin, and cytoplasm containing course, round granules that areuniform in size and stainable by eosin. Hypereosinophilia ischaracterized by an increased number of eosinophils, often associatedwith allergies, asthmas and infections.

Some use of oligonucleotides directed against specific nucleic acidsequences coding for receptors, in order to inhibit inflammatoryreactions is known. PCT Application No. WO 99/66037 by Renzi describesantisense oligonucleotides that are used for treating and/or preventingasthma, allergy, hypereosinophilia, general inflammation and cancer.Specifically, the oligonucleotides of Renzi are directed against nucleicacid sequences coding for a CCR3 receptor, a common sub-unit of IL-4 andIL-3 receptors, or a common sub-unit of IL-3, IL-5 and GM-CSF receptors.Among others, an antisense oligonucleotide identified as 107A(5′-GGGTCTGCAGCGGGATGGT-S′) (SEQ ID NO: 43), directed against thecommon, beta ((β) sub-unit of the IL-3, IL-5 and GM-CSF receptor, isdisclosed therein.

For potential clinical uses, antisense oligonucleotides should exhibitstability against degradation by serum and cellular nucleases, show lownon-specific binding to serum and cell proteins, exhibit enhancedrecognition of the target mRNA sequence, demonstrate cell-membranepermeability and elicited cellular nucleases when complexed withcomplementary mRNA. It is well documented that oligonucleotidescontaining natural sugars (D-ribose and D-2-deoxyribose) andphosphodiester (PO) linkages are rapidly degraded by serum andintracellular nucleases, which limit their utility as effectivetherapeutic agents. Chemical strategic modifications have been describedfor oligonucleotides in order to improve their stability and efficacy astherapeutic agents. The main chemical changes included, modification ofthe sugar moiety, the base moiety, and/or modification or replacement ofthe internucleotide phosphodiester linkage. To date the most widelystudied analogues are the phosphorothioate (PS) oligodeoxynucleotides,in which one of the non-bridging oxygen atoms in the phosphodiesterbackbone is replaced with a sulfur (Eckstein F., 1985, Ann. Rev.Biochem., 54: 367-402). Several antisense oligonucleotide generationshave been developed and used for in vitro and for in vivo studies(Goodchild J., 2004, Curr. Opin. Mol. Ther., 2004, 6:120-128; Urban E.and R. Noe C R., 2003, Farmaco. 58:243-258).

Recently, Renzi et al. described the use of 2′,6′-diaminopurine (DAP)and analogs thereof in nucleic molecules for anti-inflammatorycompositions (PCT Application No. WO 03/004511 A2). Also described inthis reference is the preparation of nucleic molecules having anincreased in vivo physiological efficiency and a reduced toxicity ascompared to oligonucleotides without DAP. Renzi et al. further teachesthat DAP substitution is particularly useful in preparingoligonucleotides directed to pulmonary/respiratory diseases such ascystic fibrosis, asthma, chronic bronchitis, chronic obstructive lungdisease, eosinophilic bronchitis, allergies, allergic rhinitis,pulmonary fibrosis, adult respiratory distress syndrome, sinusitis,respiratory syncytial virus or other viral respiratory tract infectionand cancer.

It would be desirable to have further antisense oligonueleotidesdirected against at least one specific common receptor for either Th2cytokines or receptors for mediators that attract cells that respond toTh2 cytokines, in order to inhibit the inflammatory reaction that ispresent in asthma or allergy and to inhibit neoplastic cellproliferation.

It would also be highly desirable to have further antisenseoligonucleotides directed against nucleic acid sequences coding forreceptors so that by inhibiting these receptors these oligonucleotidescould be employed in the therapy and/or prevention of asthma, allergy,hypereosinophilia, general inflammation and cancer.

SUMMARY OF THE INVENTION

The present invention provides the use of antisense oligonucleotides,directed against at least one common subunit of a cellular receptor,such as, for example, the common beta subunit for IL-3, IL-5, and GM-CSFreceptors or the chemokine receptor CCR3, in order to treat and/orprevent at least one of asthma, allergy, hypereosinophilia, generalinflammation and cancer.

In another aspect, the present invention provides antisenseoligonucleotides directed against a nucleic acid sequence coding for thecommon beta subunit of the IL-3, IL-5 and GM-CSF receptors so that byinhibiting these receptors they may be employed in the treatment and/orprevention of at least one of asthma, allergy, hypereosinophilia,general inflammation and cancer.

The present invention also provides antisense oligonucleotides directedagainst a nucleic acid sequence coding for the CCR3 receptor forchemokines so that by inhibiting this receptor they may be employed inthe treatment and/or prevention of at least one of asthma, allergy,hypereosinophilia, general inflammation and cancer.

The present invention also provides therapeutically effectivecompositions comprising at least one antisense oligonucleotide directedagainst nucleic acid sequences coding for the common beta subunit ofIL-3, IL-5, and GM-CSF, or the CCR3, receptors for the treatment and/orprevention of at least one of asthma, allergy, hypereosinophilia,general inflammation and cancer.

The present invention also provides therapeutically effectivecompositions comprising two antisense oligonucleotides each directedagainst nucleic acid sequences coding for the common beta subunit ofIL-3, IL-5, and GM-CSF, or the CCR3, receptors for an improved effect inthe treatment and/or prevention of at least one of asthma, allergy,hypereosinophilia, general inflammation and cancer.

According to another aspect, the present provides methods for treatingand/or preventing at least one of asthma, allergy, general inflammationand cancer comprising administering one or more antisenseoligonucleotides directed against at least one common subunit of acellular receptor, such as the common beta subunit for IL-3, IL-5, andGM-CSF or the CCR3, receptors.

The present invention seeks to provide antisense oligonucleotides forany of the foregoing as well as chemically modified antisenseoligonucleotides modified in known ways that have improved stability inthe body while exhibiting improved effectiveness and lower toxicity.

According to another aspect of the present invention, an antisenseoligonucleotide for treating and/or preventing at least one of asthma,allergy, hypereosinophilia, general inflammation and cancer is provided.The oligonucleotide is directed against a nucleic acid sequence codingfor a receptor selected from the group consisting of a CCR3 chemokinereceptor and a common beta-sub-unit of IL-3, IL-5 and GM-CSF receptors,and has a sequence selected from the group consisting of SEQ ID NO. 1,SEQ ID NO. 13 and SEQ ID NO. 14.

According to another aspect of the invention, use of the at least oneoligonucleotide for treating and/or preventing at least one of asthma,allergy, hypereosinophilia, general inflammation and cancer, isprovided. Preferably, oligonucleotides comprising both sequences SEQ IDNO. 13 and SEQ ID NO. 14 are used.

According to another aspect of the invention, a pharmaceuticalcomposition for treating and/or preventing at least one of asthma,allergy, hypereosinophilia, general inflammation and cancer is providedcomprising the at least one oligonucleotide in association with apharmaceutically acceptable carrier. Preferably, the at least oneoligonucleotide comprises both SEQ ID NO. 13 and SEQ ID NO. 14.

According to another aspect of the invention, a use of thepharmaceutical composition, for treating and/or preventing at least oneof asthma, allergy, hypereosinophilia, general inflammation and canceris provided.

According to another aspect of the invention, a method for treatingand/or preventing at least one of asthma, allergy, hypereosinophilia,general inflammation and cancer is provided comprising the step ofadministering an effective amount of (i) the at least oneoligonucleotide or (ii) the pharmaceutical composition comprising the atleast one oligonucleotide in association with a pharmaceuticallyacceptable carrier.

The invention herein also relates to modifications to an antisenseoligonucleotide(s) that do not significantly adversely affect theirability to reduce activity or inhibit expression of a target protein,but which may enhance this ability.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the preferred embodiments of the inventionwill become more apparent in the following detailed description in whichreference is made to the appended drawings wherein:

FIG. 1A shows the sequence alignment of three clones obtained from PCRamplification of the Cynomolgus monkey common beta-chain for IL-3, IL-5and GM-CSF, receptor genes with the corresponding human, chimpanzee,pork, rat and mouse, orthologues surrounding the human TOP004 complementsequence.

FIG. 1B shows the predicted amino acid sequences (SEQ ID NO: 38 humanprotein (pt), SEQ ID NO: 44 human nucleotide (nt); SEQ ID NO: 38 Pantroglydytes pt, SEQ ID NO: 45 Pan troglydytes nt; SEQ ID NO: 37 MacacaFascicularis pt, SEQ ID NO: 46 Macaca Fascicularis nt; SEQ ID NO: 40Cavia porcellus pt, SEQ ID NO: 47 Cavia porcellus nt; SEQ ID NO: 42Rattus norvegicus pt, SEQ ID NO: 48 Rattus norvegicus nt; SEQ ID NO: 41Mus musculus pt, SEQ ID NO: 49 Mus musculus nt) of the translated regionsurrounding TOP004 complementary segment in cloned Cynomolgus monkey,human, chimpanzee (AADA01213660-SEQ ID NO. 63), pork (U94688.1-SEQ IDNO. 60), rat (NM_(—)133555.1-SEQ ID NO. 61) and mouse(NM_(—)007780.1-SEQ ID NO. 62), common beta-chain DNA sequences.

FIG. 2A shows the reduced beta-chain (β_(c)) mRNA expression by varyingconcentrations of TOP004 in cynomolgus monkey PBMC as compared toentreated cells.

FIG. 2B shows the reduced CCR3 mRNA expression by varying concentrationsof TOP005 in cynomolgus monkey PBMC as compared to untreated cells.

FIG. 3A shows reduced beta-chain (β_(c)) cell surface protein expressionby different concentrations of TOP004 in cynomolgus monkey PBMCs ascompared to untreated cells.

FIG. 3B shows reduced CCR3 cell surface protein expression by differentconcentrations of TOP005 in cynomolgus monkey PBMCs as compared tountreated cells.

FIG. 4A shows reduced beta-chain (β_(c)) mRNA expression by varyingconcentrations of ASM8 in cynomolgus monkey PBMCs as compared tountreated cells.

FIG. 4B shows reduced CCR3 mRNA expression by varying concentrations ofASM8 in cynomolgus monkey PBMCs as compared to untreated cells.

FIG. 5 shows the effect of oligonucleotides on CCR3 mRNA expression inHL60 differentiated cells. Antisense oligonucleotide A86, directedagainst CCR3, is shown to decrease CCR3 mRNA expression when compared tocontrol and sense oligonucleotides with no effects on G3PDH expression.

FIG. 6 shows a calcium mobilization assay in oligonucleotide-treatedHL-60 cl-15 cells. The decreased mobilization in response to eotaxin inA86 treated cells is compared to the control and the senseoligonucleotides treated cells.

FIG. 7 shows the effect of oligonucleotides on the chemotactic responseof purified human eosinophils to eotaxin. Relative chemotactic responseof A86 treated eosinophils is compared to control and sense treatedcells.

FIG. 8 shows a calcium mobilization assay in oligonucleotide-treatedeosinophils in response to eotaxin. Calcium mobilization is compared ineosinophils treated with A86 and control or oligonucleotide sensetreated cells.

FIG. 9 shows the effect of TOP005 on cell surface expression of CCR3presented as percent of expression vs. controls in Eol-1 and U937 cells.

FIG. 10 shows the effect of TOP005 on mRNA expression in human PBMC.Gels showing G3PDH and CCR3 expression are shown above the bar graph.The ratio of CCR3 mRNA expression to G3PDH, normalized for controls ispresented on the bottom.

FIG. 11A shows modulation of beta-chain (β_(c)) mRNA expression in 107Atreated TF-1 cells using RT-PCR to detect the beta-chain (β_(c)) mRNA orcontrol G3PDH mRNA expression.

FIGS. 11B and 11C show the effect of sense oligonucleotide and 107Atreatment on beta-chain expression on the cell surface of TF-1 cells, asdetermined by FACS analysis.

FIG. 11D shows the effect of TOP004 on common beta-chain expression atthe mRNA and protein levels in U937 cells.

FIG. 12 shows the proliferation of TF-1 cells treated with 107A in thepresence of GM-CSF, IL-3, or IL-5.

FIG. 13A shows the modulation of eosinophil survival by 107A, assessedusing Trypan blue dye exclusion assay.

FIG. 13B shows the modulation of eosinophil survival by 107A as assessedby flow cytometric analysis using the Annexin-V-FITC and propidiumiodide protocol.

FIG. 14 shows the elution profile of the individual products of ASM8(TOP004 and TOP005) using DEAE anion exchange chromatography.

FIG. 15 shows the elution profile for ASM8 after treatment with CH₃COOHfor 3 hours and submitted to alkaline lysis prior to fractionation byDEAE anion exchange chromatography.

FIG. 16 shows the elution profile for ASM8 after treatment with CH₃COOHfor 6 hours and submitted to alkaline lysis prior to fractionation byDEAE anion exchange chromatography.

FIGS. 17A1, 17A2, 17B1 and 17B2 show the chemical stability of ASM8after storage under different temperatures and later eluted using DEAEanion exchange chromatography.

FIG. 18 shows melting curves for TOP004 and TOP005 in 1×PBS.

FIG. 19 shows a thermodynamics summary based on results of melting curvefits of TOP004 and TOP005 in 1×PBS.

FIGS. 20A and 20B show the concentrations of TOP004 and TOP005 and theirmetabolites in monkey plasma at day 1 after treatment with a high-doseof ASM8.

FIGS. 21A and 21B show the concentrations of TOP004 and TOP005 and theirmetabolites in monkey plasma at day 14 after treatment with a high-doseof ASM8.

DETAILED DESCRIPTION OF THE INVENTION

Several inflammatory mediators play a role in the appearance andperpetuation of inflammation in the airways of patients with asthma.Some mediators attract the inflammatory cells into the airways eitherthrough chemotaxis of eosinophils. Many of these chemokines act mostlyin asthmatic or allergic inflammation through the CCR3 receptor. Othermediators cause the priming and increased survival of inflammatory cellsin the airways or skin such as IL-3, IL-5, and GM-CSF. An improvement inasthma has been shown when there is a decrease in these inflammatorymediators in the airways.

Furthermore, cancer, characterized by abnormal proliferation ofimmortalized cells, can be caused by the release of inflammatorymediators and/or growth factors that act through receptors and lead tocellular proliferation. Amongst these, GM-CSF has been shown to be animportant growth factor for several tumor cells. The chemokine receptorCCR3 was characterized in malignant B lymphocytes recovered frompatients with chronic lymphocytic leukemia (CLL) and with hairy cellleukemia (HCL), (Trentin et al., 2004, Blood, 104, 502-508). Indeed, thetransactivation of EGFR through CCR3 was found a critical pathway thatelicits MAP kinase activation and cytokine production in bronchialepithelial cells (Adachi et al., 2004, Biochem. Biophys. Res. Commun.320, 292-396). The inhibition of proliferation and metastasis ofcancerous cells by blocking the receptors for growth factors or thechemokine receptor CCR3 could be important in the therapy of certaincancers.

In one embodiment of the invention, a novel antisense oligonucleotideidentified as 828 (5′-GTTACTACTTCCACCTGCCTG-3′, (SEQ ID NO. 1)) anddirected against the CCR3 chemokine receptor is provided. The examplesdisclosed herein show that 828 is effective at decreasing or blockingCCR3 mRNA expression in human cell lines.

In another embodiment of the invention, novel antisense oligonucleotidesTOP004 and TOP005 based on the previously disclosed 107A and the above828 are provided. TOP004 (5′-GGGTCTGCXGCGGGXTGGT-3′ (SEQ ID NO. 13)where X represents a DAP modification of an adenosine residue), as with107A, is a 19-mer directed against the mRNA of the common beta (β)-chainof the IL-3, IL-5, and GM-CSF receptors. TOP005 (5′-GTTXCTXCTTCCXCCTGCCTG-3′ (SEQ ID NO. 14), where X represents a DAP modification of anadenosine residue), as with 828, is a 21-mer directed against the mRNAof the chemokine receptor CCR3. A composition comprising both TOP004 andTOP005 is identified as a part of ASM8.

As disclosed herein, TOP004 and TOP005 possess activity in a non-humanprimate system, thus validating the use of the cynomolgus monkey forsafety assessment. FIG. 1 shows the sequencing of the common beta-chaingene of cynomolgus monkey. The cynomolgus beta-chain sequencecomplementary to TOP 004 showed significant homology. The very highdegree of identity between the monkey and the human beta-chain sequencesuggest a probable functional activity of TOP004 in cynomolgus monkey.The effectiveness of TOP 004 and TOP 005 at blocking or decreasingexpression of the common beta-chain and CCR3 in monkey peripheral bloodmononuclear cells is shown in FIGS. 2, 3, and 4. The results show thatboth of TOP004 and TOP005 directed against human gene targets areeffective at reducing expression of their respective targets incynomolgus monkey peripheral blood mononuclear cells (PBMC). ASM8,containing both TOP004 and TOP005 significantly inhibited both thecommon beta-chain expression and CCR3, receptors, either to a greaterdegree or to the same extent at a lower concentration. TOP004 and TOP005together therefore exhibit synergistic effects in blocking beta-chainand CCR3 mRNA expression. Furthermore, in Tables 7 and 8, trachealsamples, taken from monkeys treated with ASM8, were analyzed for thelevel of mRNA expression. The expression of the target genes wasnormalized to the mRNA levels for inflammatory cytokines (IL-4 andTNF-α). Even approximately 24 hours after administration of ASM8, therelative expression of the β_(c)-subunit and CCR3 mRNA to IL-4 mRNA wasdecreased by 29% and 24%, respectively, and the expression relative toTNF-α was decreased by 30% and 24%, respectively, in ASM8-treatedanimals.

In FIGS. 5-10, antisense nucleotides, including A86 and TOP005, directedagainst the CCR3 mRNA were tested for efficacy in human cells and celllines. When assessed by semi-quantitative reversetranscription-polymerase chain reaction (“RT-PCR”), the antisenseoligonucleotides caused inhibition of CCR3 mRNA expression. Further,using FACS analysis, it is shown that cell surface expression of CCR3protein was inhibited by antisense oligonucleotide treatment as well.Moreover, the functional inhibition of CCR3 was confirmed by inhibitionof calcium (Ca⁺⁺) mobilization in purified eosinophils after stimulationwith eotaxin. In addition, the oligonucleotides inhibited eosinophilchemotaxis by 55% in a chemotaxis assay.

In FIGS. 11-13, 107A and TOP004 antisense were used to treat variouscells. TF-1 cells incubated with 107A showed reduced beta-chain mRNAexpression. 107A also inhibited TF-1 cell proliferation in the presenceof IL-3, IL-5, or GM-CSF. Furthermore, 107A reduced, in a dose-dependentmanner, the anti-apoptotic effect of IL-5 on eosinophils. U937 cellsincubated with TOP004 showed reduced common beta-chain expression at thelevel of mRNA and protein. The antisenses 107A and TOP004 were thushighly effective in blocking the expression of beta-chain mRNA, proteinand functional at blocking the associated cellular responses in humancell cultures.

In FIGS. 14-17B2, the stability of ASM8 is shown by eluting thecomposition under varying conditions. ASM8 was eluted using the DEAEanion exchange high performance liquid chromatography (HPLC)-basedfractionation system to assess the integrity of ASM8 and its degradationproducts after storage at different temperatures. ASM8 components didnot undergo any detectable degradation when stored at −20° C., 4° C.,30° C., or 40° C. for up to 2 months.

In FIGS. 18-19, melting curves and thermodynamic summaries provided forASM8 show that the two oligonucleotide strands do not interactsignificantly in solution.

In FIGS. 20-21, the concentrations of ASM8 oligonucleotide constituentsand their primary metabolites (n−1) in monkey plasma samples weremeasured. The samples were collected during a nonclinical toxicity trialin which the animals were treated for 14 consecutive days viainhalation.

Antisense oligonucleotides directed against the common beta subunit ofIL-3, TL-5 and GM-CSF, and the CCR3, receptors, and against nucleicacids coding therefore, are thus provided. Pharmaceutical compositionscomprising the oligonucleotides with a pharmaceutically acceptablecarrier are also provided. Uses of the oligonucleotides and methodscomprising administering the oligonucleotides for treating and/orpreventing at least one of asthma, allergy, hypereosinophilia, generalinflammation and cancer are described.

The terms “nucleic acid” and “nucleic acid molecule” as usedinterchangeably herein, refer to a molecule comprised of nucleotides,i.e., ribonucleotides, deoxyribonucleotides, or both. The term includesmonomers and polymers of ribonucleotides and deoxyribonucleotides, withthe ribonucleotide and/or deoxyribonucleotides being connected together,in the case of the polymers, via 5′ to 3′ linkages. However, linkagesmay include any of the linkages known in the nucleic acid synthesis artincluding, for example, nucleic acids comprising 5′ to 2′ linkages. Thenucleotides used in the nucleic acid molecule may be naturally occurringor may be synthetically produced analogues that are capable of formingbase-pair relationships with naturally occurring base pairs.

Examples of non-naturally occurring bases that are capable of formingbase-pairing relationships include, but are not limited to, aza anddeaza pyrimidine analogues, aza and deaza purine analogues, and otherheterocyclic base analogues, wherein one or more of the carbon andnitrogen atoms of the purine and pyrimidine rings have been substitutedby heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and thelike.

The term “nucleic acid backbone” as used herein refers to the structureof the chemical moiety linking nucleotides in a molecule. This mayinclude structures formed from any and all means of chemically linkingnucleotides. A modified backbone as used herein includes modificationsto the chemical linkage between nucleotides, as well as othermodifications that may be used to enhance stability and affinity, suchas modifications to the sugar structure. For example an [alpha]-anomerof deoxyribose may be used, where the base is inverted with respect tothe natural [beta]-anomer. In a preferred embodiment, the 2′-OH of thesugar group may be altered to 2′-O-alkyl or 2′-O-alkyl-n(O-alkyl), whichprovides resistance to degradation without comprising affinity.

The term “oligonucleotide” as used herein refers to a nucleic acidmolecule comprising from about 1 to about 100 nucleotides, morepreferably from 1 to 80 nucleotides, and even more preferably from about4 to about 35 nucleotides.

Antisense oligonucleotide compounds in accordance with the presentinvention also include siRNAs (small interfering RNAs) and the RISCs(RNA-induced silencing complexes) containing them that result from theRNAi (RNA interference) approach. The RNA interference (RNAi) approach,which has been described recently, is considered as a new tool for theinhibition of target gene expression. As already known some years ago,RNAi is based on an ancient anti-viral defence mechanism in lowereukaryotes. It is induced by double-stranded RNA and its processing to21-23 nt small interfering RNAs (siRNAs), which cause the degradation ofhomologous endogenous mRNA after hybridizing to the target mRNA in asingle stranded fashion with the assistance of the RISC complex. The wayRNAi works is still to be fully elucidated, but it already serves as afirst-choice approach to generate loss-of-function phenotypes among abroad variety of eukaryotic species, such as nematodes, flies, plants,fungi and mammals.

Antisense oligonucleotide compounds in accordance with the presentinvention also include ribozymes and short nucleotide sequences, singleor double stranded, RNA or DNA, which may incorporate chemicalmodifications as described above, capable of inhibiting genetranscription and/or translation in vitro and/or in vivo.

The term “modified oligonucleotide” and “modified nucleic acid molecule”includes antisense oligonucleotide compounds that have been modifiedwithout significant adverse effect to their activity, for example, bythe insertion or deletion of 1 or more bases. In particular, theaddition or deletion of bases at the terminal ends of theoligonucleotides that exhibit 100% complementation to the gene they aredirected against can generally be made without significant loss ofinhibitory activity. Such modifications may be made in order to increaseactivity or to provide enhanced stability of the oligonucleotide. Inaddition, substitution of 1 or more bases in the present antisenseoligonucleotide compounds may also be made without adverse effect toactivity, for example, substitution of purine with another purine(adenine, guanine) and pyrimidine with pyrimidine (cytosine, thymine,uracil). Modified oligonucleotide and modified nucleic acid molecule asused herein also include nucleic acids, including oligonucleotides, withone or more chemical modifications at the molecular level of the naturalmolecular structures of all or any of the nucleic acid bases, sugarmoieties, internucleoside phosphate linkages, as well as moleculeshaving added substituents, such as diamines, cholesteryl or otherlipophilic groups, or a combination of modifications at these sites. Theinternucleoside phosphate linkages can be phosphodiester,phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, phosphorothioate,methylphosphonate, phosphorodithioate, bridged phosphorothioate and/orsulfone internucleotide linkages, or 3′-3′,2′-5′ or 5′-5′ linkages, andcombinations of such similar linkages (to produce mixed backbonemodified oligonucleotides). The modifications can be internal (single orrepeated) or at the end(s) of the oligonucleotide molecule and caninclude additions to the molecule of the internucleoside phosphatelinkages, such as cholesteryl, diamine compounds with varying numbers ofcarbon residues between amino groups and terminal ribose, deoxyriboseand phosphate modifications which cleave or cross-link to the oppositechains or to associated enzymes or other proteins. Electrophilic groupssuch as ribose-dialdehyde may be covalently linked with an epsilon aminogroup of the lysyl-residue of such a protein. A nucleophilic group suchas n-ethylmaleimide tethered to an oligomer could covalently attach tothe 5′ end of an mRNA or to another electrophilic site. The termmodified oligonucleotides also includes oligonucleotides comprisingmodifications to the sugar moieties such as 2′-substitutedribonucleotides, or deoxyribonucleotide ionomers, any of which areconnected together via 5′ to 3′ linkages. Modified oligonucleotides mayalso be comprised of PNA or morpholino modified backbones where targetspecificity of the sequence is maintained. The term modifiedoligonucleotides also includes oligonucleotide compounds, as definedherein, of a form that does not significantly adversely affect theiractivity to reduce activity or inhibit expression of a target protein,but which may enhance this activity.

Modified oligonucleotides also include oligonucleotides that are basedon or constructed from arabinonucleotide or modified arabinonucleotideresidues, including but not limited to antisense oligonucleotideconstructs based on beta-arabinofuranose and its analogues.Aribonucleosides are stereoisomers of ribonucleosides, differing only inthe configuration at the 2′-position of the sugar ring. PCT ApplicationNo. WO 99/67378 by Damha et al. (1), which is hereby incorporated byreference in its entirety, discloses arabinonucleic acids (ANA)oligomers and their analogues for improved sequence specific inhibitionof gene expression via association to complementary messenger RNA. Dahmaet al. further teaches sugar-modified oligonucleotides that form aduplex with its target RNA sequence resulting in a substrate for RNaseH.Specifically, oligomers comprising beta-D-arabinonucleotides and2′-deoxy-2′-fluoro-beta-D-arabinionucleosides are disclosed. PCTApplication No. WO 02/20773 also by Dahma et al. (2), which is herebyincorporated by reference in its entirety, discloses oligonucleotidechimeras used to inhibit gene transcription and expression in a sequencespecific manner. Specifically, Dahma et al. (2) teaches antisenseoligonucleotides constructed from arabinonucleotides flanking a seriesof deoxyribose nucleotide residues of variable length. Antisenseoligonucleotides so constructed are used to hybridize and inducecleavage of complementary RNA. PCT Application No. WO 03/037909 also byDahma et al. (3), which is hereby incorporated by reference in itsentirety, discloses oligonucleotides having an internal acyclic linkerresidue. Antisense oligonucleotides prepared with an acyclic linker areused to prevent or deplete function of a target nucleic acid of interestsuch RNA. PCT Application No. WO 03/064441 also by Dahma et al. (4),which is hereby incorporated by reference in its entirety, disclosesoligonucleotides having alternating segments of sugar-modifiednucleosides and 2′deoxynucleosides and also alternating segments ofsugar-modified nucleotides and 2′deoxynucleotides. Antisenseoligonucleotides having these alternating segments are disclosed to beused to prevent or deplete function of a target nucleic acid of interestsuch as RNA.

The term “substantially nuclease resistant” refers to nucleic acids thatare resistant to nuclease degradation, as compared to naturallyoccurring or unmodified nucleic acids. Modified nucleic acids of theinvention are at least 1.25 times more resistant to nuclease degradationthan their unmodified counterpart, more preferably at least 2 times moreresistant, even more preferably at least 5 times more resistant, andmost preferably at least 10 times more resistant than their unmodifiedcounterpart. Such substantially nuclease resistant nucleic acidsinclude, but are not limited to, nucleic acids with modified backbonessuch as phosphorothioates, methylphosphonates, ethylphosphotriesters,2′-0-methylphosphorothioates, 2′-O-methyl-p-ethoxy ribonucleotides,2′-O-alkyls, 2′-O-alkyl-n(O-alkyl), 3′-O-alkyls, 3′-O-alkyl-n(O-alkyl),2′-fluoros, 2′-deoxy-erythropentofuranosyls, 2′-O-methylribonucleosides, methyl carbamates, methyl carbonates, inverted bases(e.g., inverted T's), or chimeric versions of these backbones.

The terms “CCR3 and common beta-chain for IL-3/IL-5/GM-CSF, -receptors,antisense oligonucleotides” as used herein each refer to anoligonucleotide that is targeted, respectively, to sequences that affectCCR3 chemokine receptor and the common beta-chain for IL-3/IL-5/GM-CSF,-receptors, expression and/or activity. These include, but are notlimited to, CCR3 chemokine receptor and the common beta-chain forIL-3/IL-5/GM-CSF, -receptors, DNA coding sequences, DNA promotersequences, DNA enhancer sequences, mRNA encoding sequences, and thelike.

As discussed above, one embodiment of the present invention providesantisense oligonucleotides targeted to sequences that affect CCR3chemokine receptor and the common beta-chain for IL-3/IL-5/GM-CSF,-receptors, expression and/or activity. In one embodiment the antisenseoligonucleotide may comprise fragments or variants of these sequences,as will be understood by a person skilled in the art, that may alter theoligonucleotide make-up and/or length, but which maintains or increasesthe activity of the oligonucleotide to down-regulate gene expression. Inanother embodiment the present invention provides for combinations of atleast two antisense oligonucleotides from the sequences identified asSEQ ID NO.1, SEQ ID NO.13 and SEQ ID NO.14.

The terms “treatment”, “treating”, “therapy” and the like are usedherein to generally mean obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete cure for a diseaseand/or amelioration of an adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in asubject as previously defined, particularly a human, and includes:

(a) preventing a disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(b) inhibiting a disease, i.e., arresting its development; or

(c) relieving a disease, i.e., causing regression of the disease.

The term “pharmaceutically acceptable” as it is used herein with respectto carriers, surfactants and compositions refers to substances which areacceptable for use in the treatment of a subject patient that are nottoxic or otherwise unacceptable for administration by any of the routesherein described.

The invention is generally directed toward the treatment of subjects bythe administration of therapeutically effective amounts of antisenseoligonucleotide compounds in accordance with the present invention,including siRNA, ribozymes, short nucleotide sequences as single ordouble stranded including RNA and/or DNA that may be complementary to atarget nucleic acid, or may optionally be modified as described above,an RNA oligonucleotide having at least a portion of said RNAoligonucleotide capable of hybridizing with RNA to form anoligonucleotide-RNA duplex, or a chimeric oligonucleotide, that willdownregulate or inhibit the expression of an endogenous gene in vivo.

By “therapeutically effective” amount is meant a nontoxic but sufficientamount of an antisense oligonucleotide compound to provide the desiredtherapeutic effect. In the present case, that dose of antisenseoligonucleotide compound effective to relieve, ameliorate, or preventsymptoms of the condition or disease being treated, e.g. diseaseassociated with allergy, asthma, inflammatory disease such asinflammatory respiratory disease.

The term “allergy” as used herein, describes any undesirable immuneresponse by the body to a substance to which it has becomehypersensitive.

The formulations of the present invention are preferably administereddirectly to the site of action and thus preferably are topical,including but not limited to, oral, intrabuccal, intrapulmonary, rectal,intrauterine, intratumor, nasal, intrathecal, inhalable, transdermal,intradermal, intracavitary, iontophoretic, ocular, vaginal,intraarticular, otical, transmucosal, rectal, slow release or entericcoating formulations. Without limiting any of the foregoing,formulations of the present invention may also be intracranial,intramuscular, subcutaneous, intravascular, intraglandular, intraorgan,intralymphatic, intraperitoneal, intravenous, and implantable. Thecarriers used in the formulations may be, for example, solid and/orliquid carriers.

Reference may be made to “Remington's Pharmaceutical Sciences”, 17thEd., Mack Publishing Company, Easton, Pa., 1985, for other carriers thatwould be suitable for combination with the present oligonucleotidecompounds to render compositions/formulations suitable foradministration to treat respiratory disease.

Optionally, the presently described oligonucleotides may be formulatedwith a variety of physiological carrier molecules. The presentlydescribed oligonucleotides may also be complexed with molecules thatenhance their ability to enter the target cells. Examples of suchmolecules include, but are not limited to, carbohydrates, polyamines,amino acids, peptides, lipids, and molecules vital to cell growth. Forexample, the oligonucleotides may be combined with a lipid, theresulting oligonucleotide/lipid emulsion, or liposomal suspension may,inter alia, effectively increase the in vivo half-life of theoligonucleotide.

The pharmaceutical compositions provided herein may comprise antisenseoligonucleotide compounds described above and one or morepharmaceutically acceptable surfactants. Suitable surfactants orsurfactant components for enhancing the uptake of the antisenseoligonucleotides of the invention have been previously described in U.S.Application Publication No. 2003/0087845, the contents of which areincorporated herein with respect to surfactants The application statesthat suitable surfactants “ . . . include synthetic and natural as wellas full and truncated forms of surfactant protein A, surfactant proteinB, surfactant protein C, surfactant protein D and surfactant protein E,di-saturated phosphatidylcholine (other than dipalmitoyl),dipalmitoylphosphatidylcholine, phosphatidylcholine,phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine; phosphatidic acid, ubiquinones,lysophosphatidylethanolamine, lysophosphatidylcholine,palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols,sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate,glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate,cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, cholinephosphate; as well as natural and artificial lamelar bodies which arethe natural carrier vehicles for the components of surfactant, omega-3fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid,non-ionic block copolymers of ethylene or propylene oxides,polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomericand polymeric, poly (vinyl amine) with dextran and/or alkanoyl sidechains, Brij 35™, Triton X-100™ and synthetic surfactants ALEC™,Exosurf™, Survan™ and Atovaquone™, among others. These surfactants maybe used either as single or part of a multiple component surfactant in aformulation, or as covalently bound additions to the 5′ and/or 3′ endsof the antisense oligonucleotides.”

The antisense component of the present compositions may be contained ina pharmaceutical formulation within a lipid particle or vesicle, such asa liposome or microcrystal. As described in U.S. Pat. No. 6,025,339, thelipid particles may be of any suitable structure, such as unilamellar orplurilamellar, so long as the antisense oligonucleotide is containedtherein. Positively charged lipids such as N-[1-(2,3-diolcoyloxi)propyl]-N,N,N-trimethyl-ammoniumethylsulfate, or “DOTAP,” areparticularly preferred for such particles and vesicles. The preparationof such lipid particles is well known. See, e.g., U.S. Pat. No.4,880,635 to Janoff et al.; U.S. Pat. No. 4,906,477 to Kurono et al.;U.S. Pat. No. 4,911,928 to Wallach; U.S. Pat. No. 4,917,951 to Wallach;U.S. Pat. No. 4,920,016 to Allen et al.; U.S. Pat. No. 4,921,757 toWheatley et al.; etc.

The composition of the invention may be administered by any means thattransports the antisense oligonucleotide compound to the desired site,such as for example, the lung. The antisense compounds disclosed hereinmay be administered to the lungs of a patient by any suitable means, butare preferably administered by inhalation of an aerosol comprised ofrespirable particles that comprise the antisense compound.

The composition of the present invention may be administered into therespiratory system as a formulation including particles of respirablesize, e.g. particles of a size sufficiently small to pass through thenose, mouth and larynx upon inhalation and through the bronchi andalveoli of the lungs. In general, respirable particles range from about0.5 to 10 microns in size. Particles of non-respirable size that areincluded in the aerosol tend to deposit in the throat and be swallowed,and the quantity of non-respirable particles in the aerosol ispreferably thus minimized. For nasal administration, a particle size inthe range of 10-500 micro-M (micro-meter)) is preferred to ensureretention in the nasal cavity.

Liquid pharmaceutical compositions of active compound (the antisenseoligonucleotide compound(s)) for producing an aerosol may be prepared bycombining the antisense compound with a suitable vehicle, such assterile pyrogen free water or phosphate buffered saline.

A solid particulate composition comprising the antisense compound mayoptionally contain a dispersant that serves to facilitate the formationof an aerosol as well as other therapeutic compounds. A suitabledispersant is lactose, which may be blended with the antisense compoundin any suitable ratio, e.g., a 1 to 1 ratio by weight.

The antisense compositions may be administered in ananti-bronchoconstriction, anti-allergy(ies) and/or anti-inflammatoryeffective amount, which amount depends upon the degree of disease beingtreated, the condition of the subject patient, the particularformulation, the route of administration, the timing of administrationto a subject, etc. In general, intracellular concentrations of theoligonucleotide of from 0.05 to 50 microM, or more particularly 0.2 to 5microM, are desirable. For administration to a mammalian patient such asa human, a dosage of about 0.001, 0.01, 0.1, or 1 mg/Kg up to about 50,or 100 mg/Kg or more is typically employed. However, other doses arealso contemplated. Depending on the solubility of the active compound inany particular formulation, the daily dose may be divided among one orseveral unit dose administrations.

The aerosols of liquid particles comprising the antisense compound maybe produced by any suitable means, such as with a nebulizer. Nebulizersare commercially available devices that transform solutions orsuspensions of the active ingredient into a therapeutic aerosol misteither by means of acceleration of a compressed gas, typically air oroxygen, through a narrow venturi orifice or by means of ultrasonicagitation. Suitable formulations for use in nebulizers comprise theactive antisense oligonucleotide ingredient in a liquid carrier in anamount of up to 40% w/w preferably less than 20% w/w of the formulation.The carrier is typically water or a dilute aqueous alcoholic solution,preferably made isotonic with body fluids by the addition of, forexample, sodium chloride. Optional additives include preservatives ifthe formulation is not prepared sterile, for example, methylhydroxybenzoate, anti-oxidants, anti-bacterials, flavorings, volatileoils, buffering agents and emulsifiers and other formulationsurfactants.

The aerosols of solid particles comprising the active oligonucleotidecompound(s) and a pharmaceutically acceptable surfactant may likewise beproduced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles that are respirable, as explained above, andgenerate a volume of aerosol containing a predetermined metered dose ofa medicament at a rate suitable for human administration. The activeoligonucleotide ingredient typically comprises from 0.1 to 100 w/w ofthe formulation. A second type of illustrative aerosol generatorcomprises a metered dose inhaler. Metered dose inhalers are pressurizedaerosol dispensers, typically containing a suspension or solutionformulation of the active ingredient in a liquified propellant. Duringuse these devices discharge the formulation through a valve adapted todeliver a metered volume, typically from 10 to 150 microL, to produce afine particle spray containing the active ingredient. Suitablepropellants include certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or hydrofluoroalkanes and mixtures thereof.The formulation may additionally contain one or more co-solvents, forexample, ethanol, emulsifiers and other formulation surfactants, such asoleic acid or sorbitan trioleate, anti-oxidants and suitable flavoringagents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 1 to 150liters per minute.

In a further aspect of the present invention, an article of manufactureis provided which includes packaging material contained within which isa pharmaceutically acceptable antisense oligonucleotide composition thatis therapeutically effective to treat conditions associated withallergy, asthma, rhinitis and inflammatory disease. In one embodiment,the composition comprises an antisense oligonucleotide compound that iseffective to inhibit a CCR3 chemokine receptor or the common beta-chainfor IL-3/IL-5/GM-CSF, -receptors, gene, said oligonucleotide compoundbeing at least 50% complementary to the gene. In another aspect, thecomposition comprises at least 2 antisense oligonucleotide compounds,each antisense oligonucleotide compound being capable of down-regulatingthe CCR3 chemokine receptor and the common beta-chain forIL-3/IL-5/GM-CSF, -receptors, gene, each antisense oligonucleotidecompound being present at a concentration at which the antisenseoligonucleotide compound is practically ineffective on its own todownregulate the gene it is directed against, the combination of theantisense oligonucleotide compounds being effective to downregulate atleast one of the genes that the antisense oligonucleotides are directedagainst.

In one embodiment, the packaging material of the article comprises alabel which indicates that the composition can be used to treatinflammatory respiratory disease and may additionally include anindication that the disease is one of allergy, rhinitis and asthma.

In another embodiment, the packaging material of the article comprises alabel which indicates that the composition can be used to treatinflammatory respiratory disease, and may additionally include anindication that the disease is one of allergy, asthma,hypereosinophilia, bronchitis, rhinitis or sinusitis.

For the purposes of the present invention, the packaging material may beany suitable material for packaging a nucleotide-containing compositionin accordance with the present invention, including a bottle or othercontainer (either plastic or glass), a carton, a tube, or otherprotective wrapping. As will be appreciated, the packaging may vary withthe nature of the oligonucleotide composition, for example, a liquidformulation may be packaged differently than an aerosol formulation.

The present invention will be more readily understood by referring tothe examples that are given to illustrate the following invention ratherthan to limit its scope. With respect to these examples, the followingwere methods and materials were used.

EXAMPLES Materials and Methods

Materials

The following materials and reagents were used for the experiments: RPMI1640 (Wisent, cat# 10040 CV); FBS (Fetal Bovine Serum, Wisent,cat#80150); Penicillin-Streptomycin (GIBCO, cat#15140-122); HEPES(Wisent, cat#26060CI); L-glutamine (Gibco, cat#25030-081); SodiumPyruvate (Wisent, cat#25000-Ci); PBS Sterile (GIBCO, cat#25030-081);Hanks Balanced Salt Solution (HBSS, cellgro, cat#20021-cv); SuperscriptFirst-Strand Synthesis System for RT-PCR kit (Invitrogen,cat#11904-018); dNTPs (Invitrogen, cat#10297-018; oligo(dT)₁₂₋₁₈(Invitrogen, cat#11904-018); Qiagen RNAeasy Mini Kit (Qiagen,cat#74106); Qiagen gel extraction kit (Qiagen, cat#28704); Qiagen PCRextraction kit (Qiagen, cat#28104); β-Mercaptoethanol (Sigma,cat#M-6250); 99% Ethanol (Commercial alcohols Inc., Brampton, Ontario,Canada); QiaVac 24 Manifold (Qiagen, cat#19403); DisposableVacconnectors (Qiagen, cat#19407); DNase I kit (Fermentas',cat.#EN0521); RiboGreen Quantification Reagent (Invitrogen-Molecularprobes, cat#R-11490); Taq PCR core kit (Qiagen, cat#201223), Hema-3stain set (Fisher scientific Co. cat#122-911, lot#999901); Alamar Blue(Biosource cat#DAL1100); Human CCR3 primer pair (R&D systems, cat#RDP-209-025); Human GAPDH primer pair (R&D systems, cat# RDP-39-025);Ficoll (Amersham Biosciences; cat#: 17-1440-03); anti-CD16 (Eosinophilspurification kit, Miltenyi Biotec, Auburn, Calif., cat#130-045-701);rhEotaxin (Biosource, cat# PMC1434); Chemotaxis chamber and membranes(NeuroProbe, Nucleopore-Neuroprobe, Cabin John, Md.); Human serumalbumin (SIGMA; cat.#A9511); Anti human IL-3/IL-5/GM-CSF Receptorsbeta-chain (mouse monoclonal IgG2b; Santa Cruz Biotechnology,cat.#sc-457); Anti-mouse IgG2b (goat monoclonal, Alexa Fluor 488,Molecular Probes, cat#A-21141); Anti-human CCR3 antibody (ratmonoclonal, IgG2a, R&D, cat.# MAB155); Anti-rat IgG (goat monoclonal,Alexa Fluor 633, Molecular Probes, cat.# A-21094); rhGM-CSF (R & Dsystems, cat#215-GM-005); rhIL-3 (R & D systems, cat#203-IL-010); rhIL-5(R & D systems, cat#205-IL-005), rhIL-2 (R&D systems, cat#202-IL-010);TOP 004-(n−1) (Biosource, Oligonucleotide with one nucleotide less on 3′end); TOP 005-(n−1) (Biosource, Oligonucleotide with one nucleotide lesson 3′ end); TOP 004-TP (Biosource, Template Probe); TOP 004-(n−1-TP-T)(Biosource, template Probe); LP (Biosource, Ligation Probe); TOP 005-TP(Biosource); TOP 004-(n−1-LP-A, Biosource); Reacti-Bindneutravidin-coated high binding capacity plates (Pierce, cat #15508); T4DNA Ligase 5 U/mL (Roche, cat #799 009); Anti-DIG-AP antibody (Roche,cat# 1093274); SuperBlock Blocking Buffer in PBS (Pierce, cat #37515);Methylumbellyferyl phosphate alkaline phosphatase substrate (MolecularProbes, cat #M-6491); Monkeys plasma samples containing ASM8; MW96 PlateWasher (Beckman Coulter); 96-well large capacity polypropylene plate,Nunc); Micropipettors from Eppendorf Research Brand; Black opaque96-well plates (Costar, cat# 3915).

Antisense Synthesis and Sequence Identification

Oligonucleotides were synthesized with a Gene Assembler-Plus™ (PharmaciaBiotech, Piscataway, N.J., USA), phosphorothioated and purified by HPLC.The TOP005 used in experiments illustrated in FIGS. 5-7 were performedwith cGMP oligonucleotides. Antisense sequences and identifications aredescribed in Table 1.

TABLE 1 Antisense Antisense Gene bank accession # # Sequence 5′-3′and/or SEQ ID NO. 828 5-GTTACTACTTCCACCTGCCTG-3 (SEQ ID NO: 1)AF224496 - SEQ ID NO. 50 773 5-TGGAAAAGCGACACCTACCTG-3 (SEQ ID NO: 2)AF247360 - SEQ ID NO. 51 786 5-CCCTTTTCCTGGAAAAGCGACA-3 (SEQ ID NO: 3)AF247360 - SEQ ID NO. 51 788 5-CTCCCTTTTCCTGGAAAAGCG-3 (SEQ ID NO: 4)AF247360 - SEQ ID NO. 51 793 5-TCCACCTCCCTTTTCCTGGA-3 (SEQ ID NO: 5)AF247361 - SEQ ID NO. 52 807 5-CCTCCTTGTTCCACCTCCCTT-3 (SEQ ID NO: 6)AF247362 - SEQ ID NO. 53 RZ1 5-ACCCATTGGCATTGCTCATTT-3 (SEQ ID NO: 7)AF247360 - SEQ ID NO. 51 RZ2 5-TCCTTGCAATTAGTGCTGCTT-3 (SEQ ID NO: 8)AF247361 - SEQ ID NO. 52 RZ3 5-TCGTGCAGTTCTTCTTTTTCA-3 (SEQ ID NO: 9)AF247362 - SEQ ID NO. 53 RZ4 5-CAGACTAGCTTCTCAGTTTTG-3 (SEQ ID NO: 10)AF247363 - SEQ ID NO. 54 RZ5 5-TGCTAATTTAGTGAAGTCCTT-3 (SEQ ID NO: 11)AF247364 - SEQ ID NO. 55 RZ6 5-CTTCTCCCTGAAAATCTCTTCT-3 (SEQ ID NO: 12)AF224495 - SEQ ID NO. 56 107A 5-GGGTCTGCAGCGGGATGGT-3 (SEQ ID NO: 43)NM_000398-1 - SEQ ID NO: 43 A86 5-CTGGGCCATCAGTGCTCTG-3 (SEQ ID NO: 29)NM_178329-1 - SEQ ID NO. 58 *TOP004 5-GGGTCTGCXGCGGGXTGGT-3(SEQ ID NO: 13) NM_000395-1 - SEQ ID NO. 59 *TOP0055-GTTXCTXCTTCCXCCTGCCTG-3 (SEQ ID NO: 14) AF224496 - SEQ ID NO. 60 *X =,X represents a DAP modification of an adenosine residue.Cells and Cell Culture

The following cell lines were used: TF-1 (Human erythroleukemia cellline, ATCC#CRL-2003); EOL-1 (Human acute myeloid “Eosinophilic” leukemiacell line; DSMZ#ACC386) and U937 (Human histicytic lymphoma cell line;ATCC#CRL-1593.2). EOL-1 and U937, were cultured in RPMI 1640 with 2 mML-glutamine; 1.5 g/L sodium bicarbonate; 4.5 g/L glucose; 10 mM Hepes; 1mM sodium pyruvate; 10% PBS, Penicillin 100 U/mL, Streptomycin 100microg/mL. The same medium is used for TF-1 culture, except thatrhGM-CSF is added at 2 ng/mL.

HL-60 Clone 15 Cell Culture and Differentiation

HL-60 clone 15 was differentiated to Eosinophils as described by Tiffanyet al., 1998, J. Immunol. 160:1385-1392. Briefly, The promyelocytic cellline HL-60 was maintained in RPMI 1640 with L-glutamine supplementedwith 10% heat-inactivated FBS and 25 mMN-[2-hydroxyethyl]piperazine-N′-[2-hydroxypropanesulfonic acid] (SigmaChemical Co., St. Louis, Mo.), pH 7.6, at 37° C. and 5% CO₂. Cells wereinduced to differentiate to cosinophil-like phenotype by treating themwith 0.5 microM butyric acid (Sigma Chemical Co., St. Louis, Mo.) for atleast 5 days. FACS analysis was used to assess the presence of thecommon beta-chain for IL-3/IL-5/GM-CSF receptors, after cellsdifferentiation.

Cell Viability and Antisense Treatment

Cell viability was systematically assayed using Alamar Blue testfollowing the manufacturer procedure. EOL-1; TF-1; HL-60 or U937 cellswere harvested by centrifugation (5 minutes, 1500 RPM, at roomtemperature), washed with 3×HBSS and re-suspended at 1×10⁶ cells/mL inRPMI medium without serum. 1×10⁶ cells were incubated, in triplicates,for 5 minutes with an exact antisense concentration (between 0 and 20microM) in a sterile microtube. Each reaction was then transferred in 12well plates and incubated at 37° C., 5% CO₂ for 5 hours for mRNAquantification or 12 hours for protein analysis. RPMI/FBS 20% was addedto a final concentration of 10% FBS and cells were incubated at 37° C.,5% CO₂ overnight. Cells were harvested by centrifugation (5 minutes,1500 RPM, at room temperature) and washed with 1×HBSS. Controlexperiments were included and consisted of cell treatments in absence ofantisense or in presence of mismatch oligonucleotides.

Purification of Human Eosinophils

The granulocyte fraction was obtained by centrifugation of whole bloodthrough Ficoll-Hypaque gradients (1.077 g/mL at 350 g for 30 minutes) toobtain the buffy coat layer. Human eosinophils were further purified bynegative selection with anti-CD16 coated immunomagnetic microbeads at 4°C. using the magnetic cell sorting system of Miltenyi Biotec (Auburn,Calif.). The purity of eosinophil populations, estimated by Giemsastaining, was typically 92%-100%.

Purification of Human and Cynomolgus Monkey PBMC

Fresh blood from cynomolgus monkeys was obtained from ITR LaboratoriesCanada Inc. PBMC were isolated by Ficoll-Hypaque density gradientcentrifugation of EDTA K3 blood from normal donors. PBMC were plated at2×10⁶ cells/ml/well in 12 well plates in RPMI 1640 cell culture mediumsupplemented with 10% heat inactivated FBS, Penicillin 100 U/mL,Streptomycin 100 microg/mL. Cell viability was assessed using AlamarBlue and was typically 85%-95%.

Human PBMC and Eosinophils Transfection

Human PBMC were harvested by centrifugation (5 minutes, 1500 RPM, atroom temperature), washed with 3×HBSS and re-suspended at 2×10⁶ cells/mLin RPMI medium 5% serum containing 10 microg/mL PHA. 2×10⁶ cells wereincubated, in triplicates, for 5 minutes with an exact antisenseconcentration (between 0 and 20 microM) in a sterile microtube. Eachreaction was then transferred in 12 well plates and incubated at 37° C.,5% CO₂ overnight for mRNA quantification or 48 hours, or less whenstated, for protein analysis. Cells were harvested by centrifugation (5minutes, 1500 RPM, at room temperature) and washed with 1×HBSS. Controlexperiments were included and consisted of cell treatments in absence ofantisense or in presence of mismatch oligonucleotides.

Purified Human Eosinophils were harvested by centrifugation (5 minutes,1500 RPM, at room temperature), washed with 3×HBSS and re-suspended at2.5×10⁶ cells/mL in RPMI medium 10% serum containing 2 nanog/mL rhGM-CSFor rhIL-5, overnight. The day after, cells were washed twice with HBSSand re-suspended at 2.5×10⁶ cells/mL in RPMI medium 5% serum and wereincubated, in triplicates, for 5 minutes with an exact antisenseconcentration (between 0 and 20 microM) in a sterile microtube. Eachreaction was then transferred in 12 well plates and incubated at 37° C.,5% CO₂ overnight for mRNA quantification or 48 hours, or less whenstated, for protein analysis. Cells were harvested by centrifugation (5minutes, 1500 RPM, at room temperature) and washed with 1×HBSS. Controlexperiments were included and consisted of cell treatments in absence ofantisense or in presence of mismatch oligonucleotides.

Monkey PBMC Transfection

Cynomolgus Monkey PBMC were harvested by centrifugation (5 minutes, 1500RPM, at room temperature), washed with 3×HBSS and re-suspended at 2×10⁶cells/mL in RPMI medium 5% serum and 10 microg/mL PHA (or 10 nanog/mLrhIL-2, when stated). 2×10⁶ cells were incubated, in triplicates, for 5minutes with an exact antisense concentration (between 0 and 20 microM)in a sterile microtube. Each reaction was then transferred in 12 wellplates and incubated at 37° C., 5% CO₂ overnight for mRNA quantificationor 48 hours, or less when stated, for protein analysis. Cells wereharvested by centrifugation (5 minutes, 1500 RPM, at room temperature)and washed with 1×HBSS. Control experiments were included and consistedof cell treatments in absence of antisense or in presence of mismatcholigonucleotides.

Flow Cytometric Analysis

Cells were Counted and re-suspended at 1×10⁶ cells per mL. The cellswere Centrifuged at 400×g for 3 min. at 20-25° C., and the supernatantsdiscarded. Thereafter, the cell pellet was re-suspended in 50 microL ofFACS buffer (1×PBS, pH 7.2-7.4; 0.5% human albumin; 2.5% human serum)and incubated at 37° C. for 30 min. Without discarding the supernatantadd primary antibody directly to the tube and mix. Incubate at 4° C.protected from light for 1 h, (Anti human CCR-3 antibody was used at 1microg per 0.5×10⁶ cells. Anti human common beta-chain was used at 2microg per 0.5×10⁶ cells). Wash with 2 mL of FACS buffer, centrifuge at400×g for 3 min. and discard the supernatant. For isotype controls,resuspend cell pellet with 300 microL of FACSFix (1×PBS, pH 7.2-7.4; 4%paraformaldehyde), keep at 4° C. protected from light.

For CCR3 and common beta-chain labeling, resuspend cell pellet with 50microL of FACS buffer and add the secondary antibody, (Anti rat IgG2aAlexa Fluor 633 was used at 1 microg per 0.5×10⁶ cells. Anti mouse IgG2bAlexa Fluor 488 was used at 2 microg per 0.5×10⁶ cells). Incubate at 4°C. protected from light for 1 h. Wash with 2 mL of FACS buffer,centrifuge at 400×g for 3 min. and discard the supernatant. Fix labeledcells with 300 microL of FACSFix, keep at 4° C. protected from light.Data were analyzed in a BD biosciences FACS calibur and processed withthe Cell Quest program.

Calcium Mobilization Assay

Eosinophils were resuspended at 1×10⁷ cells/mL in RPMI 1640 containing10% FBS and loaded by incubating with 5 M Fura-2 AM (Molecular Probes,Eugene, Oreg., USA) at room temperature for 30 min in the dark. Thecells (1×10⁶ cells/mL) were washed three times and resuspended in salinebuffer (138 mM NaCl, 6 mM KCl, 1 mM CaCl₂, 10 mM Hepes, 5 mM glucose,and 1% BSA, pH 7.4). Each 2 mL of the cell suspension was thentransferred to a quartz cuvette, which was placed in a luminescencespectrophotometer LS50B (Perkin-Elmer, Beaconsfield, UK). Ca²⁺mobilization of the cells was measured by recording the ratio offluorescence emitted at 510 nm after sequential excitation at 340 and380 nm in response to chemokine.

Chemotaxis Assay

In vitro chemotaxis was assessed in 48-well chambers (NeuroProbe, CabinJohn, Md.) using polyvinylpyrrolidone-free polycarbonate membranes with5 mm pores (Nucleopore-Neuroprobe). Control or antisense treatedeosinophils were suspended in 1×10⁶ cells/mL in RPMI 1640 mediumcontaining 0.25% BSA. The upper and lower wells contained 50 microL and31 microL of cell suspension, respectively, with the latter suspensionsupplemented with an optimal concentration of eotaxin (80 nanog/mL).After 1 hour of incubation at 37° C. in 5% CO₂, migrated cells presentin the lower well were counted. Spontaneous migration was determined inthe absence of eotaxin and factored into results.

Monkey Antisense Treatment and Toxicity Studies

This protocol was reviewed and assessed by the Animal Care Committee(ACC) of ITR Laboratories Canada Inc. All animals were cared for inaccordance with the principles outlined in the current “Guide to theCare and Use of Experimental Animals” as published by the CanadianCouncil on Animal Care and the “Guide for the Care and Use of LaboratoryAnimals”, an NIH publication.

The toxicity of ASM8, consisting of a 1:1 mixture of twooligonucleotides (TOP 004 and TOP 005) was investigated to characterizethe toxicokinetic profile of its individual oligonucleotide components,when administered by inhalation exposure once daily for 14 consecutivedays. The reversibility of any effects of ASM8 following a 14-dayrecovery period was also assessed. Any systemic hypersensitivitycondition following 14 days of inhalation exposure to ASM8 (detectableby intra-dermal injection (ID)) was also assessed.

The vehicle control article was 0.9% sodium chloride solution forinjection USP, and was used as received. Liquid formulations of the testarticle (ASM8) for aerosolization was prepared by mixing TOP 004 and TOP005 with 0.9% sodium chloride solution for injection, USP, to achieve a1:1 mixture. The target dose solution concentrations was based on pureoligonucleotide. Therefore, a correction factor to adjust for purity wasapplied for weighing and dispensing the test article components. Thecorrection factors are 1.15 for TOP 004 and 1.24 for TOP 005. Prior tothe start of the 14-day exposure period, the amounts of each respectiveoligonucleotide required for each daily exposure was weighed out,combined (as powders) in vials designated for each day of exposure, andstored frozen at −80° C. On each day of exposure the correct vial wasremoved from frozen storage, the contents dissolved in the salinevehicle, filtered through a sterile 0.2-μm filter and the formulationwas used for that day's exposure only.

Number of animals per group and treatments are set out in Tables 2 and 3below:

TABLE 2 No. of Animals Main Phase Recovery Phase Group No TreatmentMales Females Males Females 1 Vehicle 3 3 1 1 Control 2 ASM8 Low 3 3 — —Dose 3 ASM8 Mid 3 3 — — Dose 4 ASM8 High 3 3 1 1 Dose Body Weight Range2-4 kg on Day 1 of treatment Age Range Young adults on Day 1 oftreatment

TABLE 3 ASM8 exposure concentrations and dose levels (4) Aerosolconcentration Dose of ASM8 of ASM8 Group No. Treatment (mg/kg/day), (1)(mg/L), (3) 1 Vehicle control, (2) 0 0 2 ASM8 low dose 0.05 0.00795 3ASM8 mid dose 0.25 0.0397 4 ASM8 high dose 2.5 0.3976 (1): Based on anestimated body weight of 2.5 kg. (2): Vehicle control animals wereexposed to an aerosol generated from vehicle solution at an aerosolconcentration considered to be equivalent in terms of mass to thatgenerated for high-dose group. (3): The target dose and aerosolconcentrations were based on absolute purity of the test articles, whichwere achieved by utilizing the appropriate correction factors for purityin the dose solution formulation process. (4): Achieved dose levelsduring the exposure period were estimated using the following formula:D_(L) = E_(c) × RMV × T/BW, where, D_(L) = Achieved dose levels(mg/kg/day) E_(c) = Actual concentration delivered to animals (mg/L air)RMV = Minute volume (mL/min) estimated according to the formula of Bideet al., 2000, J. App. Toxicol., 20, 273-290. as detailed: RMV (L) =0.499 × W(kg)^(0.809) T = Time, duration of daily exposure (min) BW =Mean body weight (kg) during exposure period.

This estimation of achieved dose assumed 100% deposition within therespiratory tract.

In-life observations including mortality, clinical examinations, bodyweight, food consumption, electrocardiography, ophtalmoscopy, clinicalpathology, plasma level determinations, hypersensitivity testing, wereperformed on all animals.

Upon completion of the treatment period, the animals were euthanized andsubjected to anatomic pathology tests, necroscopy, organ weights,histopathology.

Semi-quantitative RT-PCR was used to measure whether there was any ASM8inhibitory effect on the common beta-chain and the CCR3 mRNA expressionon trachea samples from the high dose treated cynomolgus monkeys 24 hrsafter ASM8 administration.

HL-ELISA for Oligonucleotide Measurements in Monkey Plasma

Monkeys blood samples (approximately 1 mL each) were collected from eachanimal on Days 1 and 14 at pre-dose, 0.5, 1, 3, 6 and 24 hourspost-dose. The blood samples were centrifuged at 4° C. to generateplasma, and the plasma was separated and frozen on dry ice untilanalyzed for determination of TOP004 and TOP005 (and proximal n−1metabolites) concentrations using hybridization/ligation ELISAquantification as say.

Standard curve solution for oligonucleotide was prepared by serialdilutions for monkey plasma samples. Usual standard curve working rangeis 125 nM to 0.007629 nM. Plasma samples were diluted appropriately formeasurement in the linear portion of the standard curve, making morethan one dilution for accurate measurement.

Each standard or plasma sample was aliquoted (200 microL) in a 96-wellpolypropylene plate in which 200 microL of appropriate template probesolution was added to the 200 microL of plasma sample and incubated at37° C. for 60 minutes. 150 microL was transferred to a NeutrAvidincoated plate in duplicate and incubate 37° C. for 30 minutes. This waswashed 4 times with washing buffer using plate washer (200 microL each).150 microL ligation probe solution was added followed by incubation atroom temperature for 120 minutes. Following incubation, the sample waswashed 2 times with washing buffer using plate washer (200 microL each)followed by washing 3 times with ddH2O using plate washer (200 microL).150 microL of 1:2000 dilution (in Super block, Peirce) of anti-DIG-APwas added followed by incubation at room temperature for 30 minutes. Thesample was washed 4 times with wash buffer using plate washer (200microL). 150 microL of 10 microM MUP reagent was then added followed byincubation at room temperature for 60 minutes. Fluorescence at355ex/485em was read.

Solutions used in HL-ELISA:

Template Probe solution (0.05 microM Template probe, 60 mM Na2HPO4 pH7.4, 0.9M NaCl, 0.24% Tween-20; 10× Ligation Buffer (0.8248M Tris-Cl pH7.5, 0.0828M MgCl2, 1.93% DTT; ATP 100 mM solution (Prepare in water andadjust to pH 7±0.5 with NaOH); Ligation Probe Solution (0.067 microMoligo in 1× Ligation Buffer, 0.025 Units/mL T4 DNA ligase, 0.05 mM ATP;Washing buffer (25 mM Tris-Cl pH 7.2, 0.15M NaCl, 0.1% Tween)

Monkey Trachea Homogenization and RNA Extraction

Monkey trachea were homogenized using a polytron PT 1200 (BrinkmannInstruments) and total RNA was extracted using the Qiagen RNAeasy minikit (Qiagen, Mississauga ON, Canada) followed by DNase I digestion.Total RNA was quantified using the Ribogreen Fluorescent Assay(Invitrogen Corporation, Burlington ON, Canada). cDNA was prepared from1-2 microg RNA using the First-Strand cDNA Synthesis Using SuperScript™II RT kit (Invitrogen Corporation, Burlington ON, Canada)

RNA Extraction, Reverse Transcription and Polymerase Chain Reaction

RNA was extracted from cell pellets according to Qiagen RNAeasy mini Kitprotocol using the QiaVac 24 manifold from Qiagen and RNA was treatedwith DNase-I according to Fermentas procedures. RNA was quantified usingthe RiboGreen reagent according to the manufacturer protocol. Otherwise,RNA was quantified using a spectrophotometer. Preparation offirst-strand cDNA was performed using the Superscript First-StrandSynthesis System for RT-PCR kit from Invitrogen, in a total reactionvolume of 20 microL. Briefly, 1-2.5 microg of RNA were first denaturedat 65° C. for 5 minutes, with 0.5 mM of each dNTPs, 0.5 microg of oligo(dT)₁₂₋₁₈ and chilled on ice for at least 1 min. The mixture wasincubated at 42° C. for 2 minutes and a second pre-mix containing 1×First-Strand Buffer, 10 mM DTT, 40 units of RNaseOUT and 40 units ofSuperScript II RT was added. Reactions were incubated at 42° C. for 10minutes, at 50° C. for 1 hour and inactivated by heating at 70° C. for15 minutes. PCR was performed with optimized quantity of cDNA (100-250nanog for CCR3 and 1-10 nanog for G3PDH) in 1×PCR buffer (10×: Tris-HCl,KCl, (NIH₄)₂SO4, 15 mM MgCl₂; pH8.7) in a total reaction volume of 50microL, 0.2 mM of each dNTPs, 8.5 pmol of each PCR primer and 2.5 unitsof Taq DNA Polymerase. The mixture was heated at 94° C. for 5 minutes,followed by 30-35 cycles, each consisting of incubation for 1 minute at94° C., 45 seconds at 60° C. and 45 seconds at 72° C. Supplementalelongation was performed at 72° C. for 10 minutes. PCR products wereanalyzed by 1.5% agarose gel electrophoresis in the presence of ethidiumbromide. Quantification of PCR products was performed using the TotalLab software (Background subtraction with Rolling Ball; Ultra Lum Inc.,Model UC4800). The PCR primers were: Human CCR3 primer pair (R&Dsystems, cat# RDP-209-025); Human GAPDH primer pair (R&D systems, cat#RDP-39-025) and primers shown in Table 4. Control experiments weresystematically included and consisted of PCR on non RT-RNA.

TABLE 4 Primer ID. Primer sequence: 5′-3′. SEQ ID NO. huBcATG.for5-ATGGTGCTGGCCCAGGG-3 15 huBcATG1.for 5-CCAGGGAGATGGTGCTGG-3 16huBc6.rev 5-CCGCTTGTAGACCACCTCAAC-3 17 huBc7.rev5-CCTTGGCTGAACAGAGACGATG-3 18 mkCCR3.for 5-TGCTCTGTGAAAAAGCCGATG-3 19mkCCR3-2.rev 5-ACCAAAAGTGACAGTCCTGGC-3 20 huBc4.for5-AAGTCAGGGTTTGAGGGCTATG-3 21 huBc4.rev 5-CAAGGGGGCAGAGACAGGTAG-3 22G3pdh.for 5-ACCACAGTCCATGCCATCAC-3 23 G3pdh.rev5-TCCACCACCCCTGTTGCTGTA-3 24Oligonucleotide Chemical Degradation

To induce degradation of TOP 004 and TOP 005 prior to analysis (in orderto ensure resolution of degradation products from the intact molecules),the following treatments were performed:

-   -   Depurination: ASM8 was resuspended in 30% CH3COOH at a final        concentration of 0.5 mg/mL, and incubated for 3, 4, or 6 hours        at room temperature. The reaction was stopped by addition of 5        volumes of water and the mixture placed at −20′ prior to        lyophilization in a Speed-Vac to remove acetic acid.    -   Cleavage: the depurinated oligonucleotides were resuspended in        0.2 M NaOH (0.5 mg/mL), incubated at 50° C. for 1 hour, and        stored at −20° C. or analyzed by HPLC.        HPLC Fractionation of TOP004 and TOP005

ASM8 was weighted, and solubilized in PBS at a concentration of 0.5mg/mL (0.25 mg/mL TOP004 and 0.25 mg/mL TOP005). HPLC gradientparameters are shown below in Table 5.

TABLE 5 HPLC gradient parameters: Flow Time (min) (ml/min) % Buffer A %Buffer B 0 1 100 0 5 1 100 0 10 1 93 7 100 1 65 35 102 1 20 80 122 1 2080 124 1 100 0 144 1 100 0 146 0.1 100 0

HPLC separation was performed with a Waters 1500 Series Binary HPLC pumpcoupled to a Waters 2487 Dual λ Absorbance detector and equipped within-line degasser, oven, and 1500 series manual injector, Reodyne 7725i.The mixture of oligonucleotides was fractionated on a Waters Protein PakDEAE 5PW anion exchange column (0.5 cm×75 cm), maintained at 60° C., anddetected by UV absorption at 260 nm. The oligonucleotide mixture(volume=25 microL) was loaded onto the column in water (buffer A: water(MilliQ grade)) and the elution was performed by progressivelyincreasing the proportion of buffer B (1 M LiClO₄, (0.22 micrometerfiltered)), resulting in an increase of ionic strength of the liquidphase, which eluted the oligonucleotide from the solid phase (column).

Under the assay conditions, 62.5 microg of either TOP 004 or TOP 005produced a measurable change>0.15 absorbance unit (AU) at 260 nm.

Oligonucleotide Storage

Aliquots of ASM8 (0.5 mg/mL) in PBS were incubated at −20° C., 4° C.,30° C., and 40° C. for 2 months. At weeks 4, and 8, the HPLC profile ofASM8 was established. The control condition was defined as the HPLCprofile of ASM8 prior to any storage time (i.e., at time zero). The HPLCsystem was driven by Breeze™ (V 3.30) software from Waters.

Oligonucleotide Melting Curves and Thermodynamic Summary Tables

TOP 004 and TOP 005 were mixed at equimolar concentrations in 1×PBS (aswell as in other buffer systems). Total oligonucleotide concentrationranged from approximately 1.2 to 8.7 mM. Standard UV thermo-denaturationmethods were conducted using a Beckman DU640 spectrophotometer with a Tmaccessory. Change in absorbance was detected at 260 nm at each degreefrom 10 to 90° C. Melting curves were fitted using MELTWIN™ 3.5 softwareto determine thermodynamic parameters. Screen pictures of melting curvesand thermodynamics summary tables were produced.

Example 1 Efficacy of Antisense Oligonucleotides Directed to the CCR3Receptor

Several antisense oligonucleotides directed to the CCR3 chemokinereceptor were analyzed for their ability to inhibit mRNA expression ofthe receptor and inhibit the function of the receptor. The CCR3antisense primary screening was performed in Eol-1 and U937 cell lines.These cells express CCR3 mRNA under the normal cell culture conditionsdescribed above. Table 6 shows antisense oligonucleotides directedagainst the human CCR3 chemokine receptor.

Referring to Table 6, antisense oligonucleotide 828 directed against theCCR-3 receptor (828: 5′-GTTACTACTTCCACCTGCCTG-3′ SEQ ID NO. 1) iseffective in inhibiting mRNA expression of the receptor as shown inTable 6.

The oligonucleotide 828 is directed against the CCR3 gene and begins 48bases after the end of exon 1 and is 21 bases long. BLAST searches wereperformed on 828 and, other than to the CCR3 receptor, the next closesthomology is reported at less than 72% homology. This is considered to beinsignificant homology for achieving the complete association of twocomplementary sequences. The specificity of 828 was assessed by using amismatch oligonucleotide (SEQ ID NO. 32). The mismatch had no effect anCCR3 mRNA or house keeping gene G3PDH used as internal control in theseexperiments. The antisense oligonucleotide 828 is therefore specific.

TABLE 6 Antisense % of CCR3 mRNA IdentificationAntisense Sequence: 5′-3′ inhibition 773 5-TGGAAAAGCGACACCTACCTG-3(SEQ ID NO: 2) 73% 828 5-GTTACTACTTCCACCTGCCTG-3 (SEQ ID NO: 1) 71% 7865-CCCTTTTCCTGGAAAAGCGACA-3 (SEQ ID NO: 3) 45% 7885-CTCCCTTTTCCTGGAAAAGCG-3 (SEQ ID NO: 4) 37% 7935-TCCACCTCCCTTTTCCTGGA-3 (SEQ ID NO: 5) 35% 8075-CCTCCTTGTTCCACCTCCCTT-3 (SEQ ID NO: 6) 31%

Example 2 Efficacy of Two DAP-Substituted Oligonucleotides in MonkeyPeripheral Blood Mononuclear Cells (PBMCs)

As discussed above, antisense oligonucleotides 107A and 828 weremodified by substituting adenosine with DAP to produce antisenseoligonucleotides TOP004 and TOP005 respectively. TOP004(5′-GGGTCTGCXGCGGGXTGGT-3′ (SEQ ID NO. 13), where X represents a DAPmodification of an adenosine residue), as with 107A, is a 19-merdirected to the common beta-chain of the IL-3, IL-5, and GM-CSFreceptors. TOP005 (5′-GTTXCTXCTTCCXCCTGCCTG-3′(SEQ ID NO. 14)), as with828, is a 21-mer directed against the chemokine receptor CCR3.

The efficacies of TOP004 and TOP005 were tested both separately and incombination. ASM8 is a composition that comprises, in part, both TOP004and TOP005. The efficacy studies were performed in monkey peripheralblood mononuclear cells (PBMCs), to validate the use of this species toexplore the potential for toxic effects arising from the pharmacologicalactivity of ASM8.

For ASM8 to be effective in the Cynomolgus monkey, sufficient homologyto their target sequences must exist. The Cynomolgus Beta-chain sequenceis not available from public databases and thus the segment encompassingthe TOP004 sequence region was cloned and sequenced. However, theactivity of TOP004 across primate species can be assessed directly in arelevant in vitro system. Specifically, a peripheral blood mononuclearcell (PBMC) preparation is a suitable system to test the functionalityof TOP 004, since the common beta-chain is found on most of themononuclear leukocyte sub-populations (T and B cells, monocytes, andmacrophages).

Sequence information for the cynomolgus monkey CCR3 receptor isavailable only for the coding region; no sequence information for theTOP005 binding region is available in the public databases. The TOP005target sequence begins 48 bases after the end of exon 1 of the humangene; this intron spans more that 20 kilo base pairs, rendering itscloning and sequencing very tedious. Reports in the literature haveshown that some segments of intron sequences are conserved between humanand monkey (Rahman et al., 2004. Genomics. 8376-84). Evolutionarystudies also show that segments of homologous intronic sequences arefound across taxa (human, whale and seal), and that these segments arefound more often near the intron-exon junctions (Hare M P and Palumbi SR., 2003 Mol Biol Evol. 20, 969-978.). Functionality of TOP005 inmonkeys was tested in a PBMC preparation in which expression of the CCR3receptor is found on T and B cells subsets.

Sequencing of the Cynomolgus Monkey Common β Chain

Analysis of common beta-chain for IL3/IL-5/GM-CSF receptor genes fromhuman, chimpanzee, pork, mouse and rat revealed a high degree of genesequence similarity among vertebrates. Primer sequences for cloning PCRwere designed. The primer sequences were derived from highly conservednucleotide sequences in human, chimpanzee, pork, mouse and rat,surrounding the TOP004 oligonucleotide region of common beta-chain gene.Table 2 shows the different primers. These primers were used to amplifyspecific products from Cynomolgus PBMC cDNA. Several PCR products wereobtained, depending on the set of primers used. A nested PCR round wasused to assess the specificity of the obtained products. The positiveamplicons were cloned and sequenced.

FIG. 1A shows the sequences of three clones (SEQ ID NO.'s 25, 26 and 27respectively) obtained from PCR amplification of the cynomolgus TOP004region aligned to the human sequence (SEQ ID NO. 28) and thecorresponding region in chimpanzee (SEQ ID NO. 33), pork (SEQ ID NO.34), rat (SEQ ID NO. 35) and mouse (SEQ ID NO. 36) nucleotide sequences.Non-homologous nucleotides are shown with lower cases while conservedregions are shown in upper case. The TOP004 region is underlined. TheCynomolgus beta-chain sequence complementary to the TOP004 region showedsignificant homology (18 of 19 bases identity) in all of the threeclones sequenced. The difference was found at position 6 (starting fromthe 5′ end of TOP 004), where both an “A” and a “G” were found (“A”being the expected base). FIG. 1B shows the alignment of proteinsequences predicted from the cloned Cynomolgus (SEQ ID NO. 37) and knownnucleotide sequences from Human (SEQ ID NO. 38), chimpanzee (emb.AADA01213660) (SEQ ID NO. 39); pork (U94688.1) (SEQ ID NO. 40); mouse(NM_(—)007780.1) (SEQ ID NO. 41) and rat (NM_(—)133555.1) (SEQ ID NO.42). The nucleotide discrepancy found at position 6 (starting from the5′ end of TOP 004 where both an “A” and a “G” were found) corresponds tothe second base of the Glutamine (Q) or Lysine (K) codon in the commonbeta-chain of available protein sequences in the public data bank. Thedata presented in FIG. 1B shows that highly evolved species contain aGlutamine residue (arrow in human, chimpanzee, pork), in the TOP004complementary region. Glutamine is encoded by 2 codons, CAA or CAG. Inlower species (mouse and rat), the Glutamine is substituted by a lysineresidue. Lysine is encoded by 2 codons, AAA or AAG. In either case, anAdenosine at the second position is conserved. As such, the Adenosineresidue is a likely candidate for the Monkey sequence to be functionalas it is in the other higher vertebrates. However, GM-CSF beta-chainpolymorphisms cannot be ruled out. Freeburn et al. discloses severalmutations in the intra-cytoplasmic region of the beta-chain receptor,which could be accounted for susceptibility to leukemia, (Freeburn etal., 1997, Exp. Hematol., 25:306-311). The sequencing data presented inFIG. 1A shows that a guanosine residue can occur at position 6 startingfrom the 5′ of the underlined TOP004 sequence. In this case, the codonwill be CGG and the protein sequence will contain an Arginine (R)residue at that position. A basic base (H, K or R) in that position isreminiscent of lower vertebrates and is unlikely the case for primates.

Despite this discrepancy, the very high degree of identity between themonkey and the human beta-chain sequence suggests functionality ofTOP004 in cynomolgus monkey.

TOP 004 and TOP005 Efficacy in Cynomolgus Monkey PBMCs

TOP 004 and TOP005 were tested individually in cynomolgus monkey PBMCsfor their ability to selectively decrease the expression of thebeta-chain and CCR3, respectively. Purified monkey PBMCs were incubatedwith different concentrations of TOP004 and TOP 005.

Referring to FIGS. 2A and 2B, results from experiments performed on morethan 10 bloods obtained from monkeys are presented in bar graph A and inbar graph B. The bar graphs show reduced beta-chain and CCR3 mRNAexpression with TOP004 (A) and TOP005 (B) in monkey PBMC. The inhibitionwas specific for TOP004 and TOP005 and not due to RNA degradation or toloss of cell viability, as evidenced by the internal control (451-bpproduct corresponding to G3PDH mRNA and cell viability test). TOP004specifically reduced the expression of the common β-chain in primarymonkey PBMCs as measured by RT-PCR (FIG. 2A). Maximum efficiency wasobtained with concentrations of 10 to 15 microM, where >50% inhibitionwas mostly observed. The inhibition of monkey beta-chain by TOP004confirmed the sequencing data (FIG. 1A) that showed a very high degreeof identity between the human and the monkey beta-chain mRNA sequences.Similarly, transfection of TOP005 into monkey PBMCs diminished theexpression of CCR3 mRNA, as measured by RT-PCR (FIG. 2B). Maximuminhibition for the CCR3 mRNA expression by TOP005 was obtained at lowerantisense oligonucleotide concentrations (0.05 to 2.5 microM) than forthe β chain (10 to 15 microM).

The inhibition of mRNA expression, as measured by RT-PCR was alsocorroborated at the protein level by flow cytometry (FACS). Monkey PBMCswere incubated for 36 hrs in growth media in the presence of variousconcentration of either TOP004 or TOP005. Flow cytometry quantificationwas done as described above. Referring to FIGS. 3A and 3B, bar graphsshow beta-chain and CCR3 cell surface expression in the presence ofTOP004 and TOP005 respectively in cynomolgus monkey PBMCs. The graphsshow that, after treatment with TOP004 or TOP005, a reduction in thepercentage of cells expressing the beta-chain and CCR3 of greater than30% was achieved at 7.5 microM and 0.5 microM, respectively, wasobserved.

TOP004 and TOP005 were also tested in combination, in a 1:1 ratio(ASM8), in Cynomolgus monkey PBMCs for their ability to selectivelydecrease the expression of the beta-chain and CCR3, respectively. MonkeyPBMCs were incubated overnight in the presence of various concentrationof ASM8 before the expression of the beta-chain and CCR3 was assessed byRT-PCR. Referring to FIGS. 4A and 4B, bar graphs representing beta-chainand CCR3 mRNA expression in the presence of ASM8 in cynomolgus monkeyPBMCs are shown for more than five (5) bloods obtained from monkeys.Significant inhibition of the beta-chain was observed withconcentrations of ASM8 ranging from 2.5 to 5 microM (FIG. 4A), which islower than the optimal concentration range giving the maximum inhibitionby TOP004 alone (between 10 to 15 microM (FIG. 2A)). These results showthat the combination of TOP004 and TOP005 antisenses providesenhanced-potency and synergy of ASM8 at blocking beta-chain expression,compared to TOP004 alone. Similarly, transfection of ASM8 into monkeyPBMCs diminished the expression of CCR3 mRNA, as measured by RT-PCR(FIG. 4B). Maximum inhibition for the CCR3 mRNA expression by TOP005 wasobtained at lower antisense oligonucleotide concentration (0.05 to 5microM) than for the beta-chain (2.5 to 5 microM). The effect for ASM8on CCR3 inhibition was not clearly concentration-dependent, this resultmay reflect that maximum inhibition (plateau) is reached at lowerconcentration for CCR3 than for the beta-chain.

In summary, sequencing of the cynomolgus common beta-chain indicated avery high degree of identity (at least 18 out of 19 bases that encompassthe TOP004 sequence). It was expected that this high degree of homologywith the human gene will allow for the sufficient hybridization ofTOP004 to the monkey beta-chain mRNA to induce antisense activity andthereby diminish its expression.

TOP 004 was transfected in purified cynomolgus PBMCs to evaluate itsability to downregulate the expression of the monkey β chain. TOP004effectively decreased the expression of β chain mRNA, measured byRT-PCR, by 30 to 70%.

Similarly, TOP005 was transfected in cynomolgus monkey PBMCs and thelevel of CCR3 expression determined by semi-quantitative RT-PCR. Theresults demonstrate that TOP005 down-regulates the expression of thecynomolgus CCR3 in a range varying between 30% and 85%.

In the same way, the transfection of either TOP004 or TOP005 in monkeyPMBCs induced a specific reduction at 0.5 microM (>30%) in the number ofcells positive for the beta-chain or CCR3, measured by flow cytometry.

ASM8 was also transfected in purified monkey PBMCs to evaluate theefficacy of the combined treatment (TOP 004 and TOP 005) to downregulatethe expression of the monkey beta-chain and CCR3, mRNA. In theseconditions, ASM8 significantly reduced the expression of the beta-chainand CCR3, measured by RT-PCR, at concentration of ASM8 as low as 0.1 to0.5 microM. This also suggests that cynomolgus monkey is an appropriatespecies in which to examine potential toxic effects due to thepharmacological activity of ASM8.

Example 3 Effect of Antisense Oligonucleotides Directed Against CCR3 inHuman Cells and Cell Lines

Further experiments were performed to assess the ability of A86 andTOP005 to inhibit CCR3 mRNA expression in HL-60 differentiatedeosinophil like cells (Lee Tiffany et al, J. Immunol 1998, 160:1385-92),U937 and Eol-1 cells as well as in peripheral blood mononuclear cells(PBMC). The ability of A86 and TOP005 to inhibit eosinophil cellmigration and calcium mobilization in both HL-60 cells and humanpurified peripheral blood eosinophils was also investigated. A86 is anantisense oligonucleotide (5′CTG GGC CAT CAG TGC TCT G 3′ (SEQ ID NO.29) that corresponds to the 87-105 nucleotide sequence of the codingregion (exon 7) of CCR3. As discussed earlier, TOP005 is 828 but withall three adenosines replaced by 2,6 diaminopurine (5′GTT XCT XCT TCCXCC TGC CTG 3′ (SEQ ID NO. 14)). The 828 complementary sequence begins48 bases after the end of exon 1 and is 21 bases long. As controls forA86, a complementary sense oligonucleotide (5′CAG AGC ACT GAT GGC CCA G3′ (SEQ ID NO. 30)) and a mismatch (5′CGT GGC ACT CAG TGT CCT G 3′ (SEQID NO. 31)) were used. As a control for 828/TOP005, a mismatch (5′CCTTTG ACC TGC CAA TGC TCT 3′ (SEQ ID NO. 32)) was used.

Effect of A86 Antisense Oligonucleotides on the CCR3 in mRNA ExpressionHL-60 Clone 15-Derived Eosinophils

It is known that the clone 15 variant of HL-60 cells can be induced bybutyric acid treatment to differentiate into cells having manycharacteristics of peripheral blood eosinophils (Lee Tiffany et al, J.Immunol. 1998, 160:1385-92). Using the same differentiation protocol, weconfirmed the expression of CCR3 mRNA in differentiated HL-60 cells.RT-PCR was then performed to examine the abilities of syntheticoligonucleotides to modulate the expression of mRNA coding for the CCR3receptor in HL-60 cells differentiated into eosinophils. After cellstreatment with 10 microM of A86, CCR3 mRNA was assessed bysemi-quantitative PCR using as internal control G3PDH. Total RNA wasisolated from freshly harvested cells as described above. Referring toFIG. 5, the effect of antisense oligonucleotides against CCR3 on CCR3mRNA expression in HL60 differentiated cells is shown. In contrast tosense oligonucleotides, and mismatch oligonucleotides, antisenseoligonueleotides inhibit markedly the expression of CCR3 mRNA. Theexpression of CCR3 mRNA in cells treated with sense oligonucleotides andmismatch oligonucleotides was not significantly different from thatobtained in non-treated cells. Moreover, all oligonucleotides at theconcentration used did not affect G3PDH mRNA expression. Antisenseoligonucleotide A86 used in this experiment is therefore able to inhibitspecifically CCR3 mRNA expression.

Effect of A86 on CCR3 Protein Cell Surface Expression

It was further investigated whether the decrease mRNA for CCR3 couldreflect that of the CCR3 cell surface protein density. In this respect,flow cytometric analysis was performed to assess the expression of CCR3receptor on HL-60 derived eosinophils after treatment witholigonucleotides. After butyric acid treatment, the percentage of HL-60derived eosinophils expressing CCR3 receptor was 40%. When treated withsense and mismatch oligonucleotides (10 microM), the percentage ofpositive cells were slightly and non-significantly decreased; thepercentage of positive cells was 35% and 38% respectively. However, thedensity of CCR3 receptor on cells treated with A86 was significantlyreduced (26% of positive cells versus 40% in non-treated cells). A86 at10 microM is able to reduce CCR3 cell surface expression by 65%. Theeffect of A86 was more significant at higher concentrations.Specifically, 20 and 30 microM of A86 was used and the results show thatCCR3 cell surface expression was decreased by 75% and 85% respectively.A86, an antisense oligonucleotide to CCR3, is able to inhibit CCR3 cellsurface expression in a dose dependent manner.

Effect of A86 on Eotaxin Induced Calcium Mobilization in HL-60 Cells

A rapid transient flux of calcium is typically observed when leucocytesare stimulated by chemokines for which they express a specific receptor.This calcium mobilization can be followed in real time by Fura-2AMloaded cells and is a convenient measure of receptor activation. Thechemokine Eotaxin is a specific ligand for CCR3 receptor and induces arapid calcium influx and leukocytes chemotaxis upon ligation to thereceptor. Referring to FIG. 6, the effect of A86 on CCR3 activation isshown. Calcium mobilization in response to eotaxin was decreased in A86treated cells when compared to control and sense oligonucleotides. Cellswere treated with A86 oligonucleotide at the concentration of 10 microM.Cells treated with sense oligonucleotide were able to respond to eotaxinas non-treated cells did. In these conditions, eotaxin induces anincrease in the intracellular concentration of Ca⁺⁺. However, in cellstreated with A86, eotaxin induced much less Ca⁺⁺ mobilization. Theresults presented here show that A86 was effective at interfering withCCR3 receptor activation in HL-60 cell line.

Treatment of Purified Human Eosinophils with Antisense OligonucleotidesInhibit their Response to Eotaxin

Referring to FIG. 7, the effect of antisense oligonucleotides onchemotactic response of purified human eosinophils to eotaxin is shown.Purified human eosinophils were incubated overnight with antisenseoligonucleotides (squares) or sense oligonucleotides (circles) at theconcentration of 10 microM, in RPMI 1640 supplemented with 5% FCS andIL-5 (1.5 ng/mL). Control cells (triangles) were incubated in the sameconditions without ODNS. Data are from a single experimentrepresentative of three and are presented as the mean number of migratedcells±SD of triplicate determinations of migrating cells per 5high-power fields. Eosinophil migration was inhibited by antisenseoligonucleotides against CCR3 and this inhibition was more significantwhen eotaxin concentration was increased. At 80 ng/mL of eotaxin,eosinophil migration was decreased by 55.6%.

FIG. 8 shows calcium mobilization in eosinophils treated with antisenseoligonucleotides. When eosinophils cells are treated with A86 (10microM), Ca⁺⁺ mobilization induced by eotaxin was also inhibited whencompared to control and sense oligonucleotides.

The results presented here show that A86 was potent at interfering witheosinophils chemotaxis to eotaxin, by down regulating the CCR3 receptor.

Efficacy of TOP005

Similar experiments were performed using TOP 005. TOP005 was chosenbecause of the efficacy of 828, results from BLAST assessment of thesequence showing that 828 had no homology to known genes, the lack ofhybridization of 828 with TOP004 (experiments performed at DNA software)and its length (permitting differentiation from TOP004 when mixedtogether and separated by anion exchange HPLC).

FIG. 9 shows the effect of TOP005 on cell surface expression of CCR3.The efficacy of TOP005 was assessed in Eol-1 and U937, cells. CCR3expression was assessed by flow cytometry 36 hours after treatment withTOP 005. Results are presented as percent of expression vs. controls inEol-1 and U937 cells. The bar graphs in FIG. 9 show that TOP005inhibited CCR3 protein expression on the surface of U937 and Eol-1cells.

FIGS. 10A and 10B show the effect of TOP005 on CCR3 mRNA expression inhuman peripheral blood mononuclear cells (PBMC). Human PBMC were eitherfreshly isolated or cultured in human interleukin-2 (10 nanog/mL for 24hours). They were then exposed to TOP005 and cultured for 18 hours. InFIG. 10A, Gels showing G3PDH and CCR3 expression are shown on the top.The ratio of CCR3 mRNA expression to G3PDH, normalized for controls ispresented on the bottom. Referring to FIG. 10B, the bar graph shows thatTOP005 is effective at decreasing PBMC CCR3 mRNA expression at doses aslow as 1 microM.

Antisense oligonucleotides A86 and TOP005 can therefore inhibit CCR3mRNA expression in Eol-1 cells (a human eosinophilic cell line), HL-60cells differentiated into eosinophils and U937 cells. Inhibition of CCR3with these oligonucleotides also decreased calcium mobilization in bothHL-60 differentiated cells and human eosinophils as well as decreasedeosinophil chemotaxis to eotaxin. Neither the corresponding senseoligonucleotides nor mismatch oligonucleotides affected the response toeotaxin.

Example 4 Efficacy of TOP004 in Reducing Expression of the Common βChain Subunit of the IL-3, IL-5 and GM-CSF Receptors and AssociatedCellular Responses in Human Cell Lines

Further experiments were performed to test the effect of 107A and TOP004on the expression of the common beta-chain subunit of the IL-3, IL-5 andGM-CSF receptors.

Modulation of Beta-Chain mRNA Expression in TF-1 and U937 Cells

Referring to FIGS. 11A, 11B and 11C, modulation of beta-chain mRNAexpression in TF-1 cells is shown. TF-1 cells were treated with 107Aantisense for 12 hours. Referring to FIG. 11A, RT-PCR was performed todetect the beta-chain mRNA and G3PDH mRNA expression in TF-1 cells.Cells were treated as follows: lane 1, control untreated; lane 2, senseoligonucleotide (10 microM); lane 3, 107A (10 microM); lane 4,mismatched oligonucleotide (10 microM). Semi-quantitative RT-PCR innon-saturating conditions was used to assess the expression ofbeta-chain and G3PDH (used as a control) mRNA. Treatment with 107A (10microM) almost completely inhibited the beta-chain expression in TF-1(FIG. 11A) and U937 treated cells (data not shown). The inhibition wasspecific for 107A and was not due to RNA degradation or to loss of cellviability, as evidenced by the internal control (450-bp productcorresponding to G3PDH mRNA) (FIG. 11A). In contrast, beta-chain mRNAexpression was not inhibited in untreated control cells or in cellstreated with sense or mismatched oligonucleotide (FIG. 11A). Thus. 107Aactivity was both specific and effective in inhibiting the expression ofbeta-chain mRNA.

Referring to FIGS. 11B and 11C, the effect of sense oligonucleotide and107A treatment on beta-chain expression on the cell surface of TF-1cells, as determined by FACS analysis, is shown. FIG. 11B shows theuntreated controls (PC) vs. sense oligonucleotide treated (S-ODN) andwhere NC represents a negative control. FIG. 11C shows cell surfaceexpression in cells treated with 107A (at varying concentrations of 5,10, and 20 microM) for 36 hours. The ability of antisenseoligonucleotides to inhibit cellular beta-chain protein expressionresulted in a corresponding lower density of beta-chain subunit on thesurface of 107A-treated cells. A monoclonal antibody (MAb) against thecommon beta-chain protein of GM-CSF/IL-3/IL-5 receptors was used,together with FACS analysis, to measure the cell surface expression ofbeta-chain protein on TF-1 cells. The level of beta-chain expression byuntreated TF-1 cells was very high and was not affected by senseoligonucleotide treatment. However, increasing concentrations of 107A(5, 10, and 20 microM) significantly reduced the level of beta-chainexpression in a dose-dependent manner (the percentage of cells testingpositive in FACS analysis decreased from 69.9% to 27.8%).

FIG. 11D shows the inhibition of the expression of the common beta-chainin U937 cells following TOP004 treatment. U937 cells were incubated inthe presence of incremental concentrations of TOP004 (0.01, 0.1, 1 and10 microM) for 12 hours in serum-free media before RT-PCR and 48 hoursbefore FACS analysis. The percentage of the common beta-chain mRNA orprotein inhibitions was determined by comparing values obtained to thatof untreated cells. The experiment was performed in triplicate and thedata represents average +/−SE. The results presented in FIG. 11Ddemonstrate that TOP004 antisense, which is the DAP containing residueshomologous to 107A antisense, is effective at inhibiting the commonbeta-chain at the mRNA and protein levels. Moreover, small amounts ofTOP004 (e.g., 1 microM) were found sufficient to knock-down thebeta-chain mRNA as well as the corresponding protein. Thus, this datafavours the efficacy of DAP chemistry and its use in pharmacologicalcompositions as described above.

Cell Survival and Functional Studies

Referring to FIG. 12, proliferation of TF-1 cells treated with 107Aantisense in the presence of GM-CSF, IL-3 or IL-5 is shown. Cells wereincubated with 107A (10 microM) for 5 hours in serum-free medium,containing 1 ng/mL GM-CSF or 3 ng/mL IL-3 or 3 ng/mL IL-5. Theincubation was terminated after 2 days, and cell proliferation wasmeasured by alamar blue assay (n=3). The results are expressed as themean of absorbance (570-595)±SD.

TF-1 cells require the cytokines GM-CSF, IL-3, or IL-5 to proliferate,and the biologic response to these cytokines involves the beta-chainsignalling pathway. Inhibition of cell surface expression of beta-chainprotein was expected to inhibit the proliferation of TF-1 cells, even inthe presence of these cytokines. 107A (10 microM) caused growthinhibition of TF-1 cells in the presence of IL-3, IL-5, or GM-CSF. Theseresults demonstrate that inhibition of beta-chain cell surface proteinexpression by 107A effectively inhibited cellular biologic responses toall three cytokines.

Eosinophils express GM-CSF, IL-3 and IL-5 receptors and play a key rolein inflammation and allergy. Eosinophils require GM-CSF, IL-3, andparticularly II-5 for their differentiation, activation, and survival(Oddera et al., 1998, Lung. 176: 237-247; Ohnishi et al., 1993, J.Allergy Clin. Immunol., 92: 607-615). The ability of antisenseoligonucleotide targeting beta-chain mRNA to inhibit eosinophil survivalin response to IL-5 was investigated. Referring to FIGS. 13A and 13B,modulation of eosinophil survival by 107A is shown.

Referring to FIG. 13A, purified human eosinophils were incubated with107A at the indicated concentrations (10, 15, and 20 microM) in RPMImedium supplemented with 5% FBS and 1.5 ng/mL IL-5 overnight. Eosinophilviability was assessed using Trypan blue dye exclusion assay. Theresults are the mean results of three experiments. Treatment with 107Aat the indicated concentrations significantly reduced eosinophilsurvival in a dose-dependent manner, to 35%±12% (10 microM), 43%±2% (15microM), and 54%±7% (20 microM) of control levels (p<0.01). Eosinophilsurvival was not significantly affected by treatment with senseoligonucleotide, as a control, at a concentration of 20 microM. Thus,107A targeting the beta-chain inhibited eosinophil survival even in thepresence of culture medium containing the specific cytokine IL-5.

Referring to FIG. 13B, purified human eosinophils were incubated for 48hours in RPMI supplemented with 5% FBS and 2 ng/mL IL-5 in the presenceor absence of 107A (15 microM). Eosinophil viability was assessed byflow cytometric analysis using the Annexin-V-FITC and propidium iodideprotocol as described in material and methods.

When eosinophils were treated with 107A, their viability was decreasedby 64%, 41% due to apoptosis. In contrast, in non-treated cells andcells treated with sense oligonucleotide, the percentage of dead cellswas lower.

Thus, 107A antisense specifically inhibits the expression of the commonbeta-chain in TF-1 cell and primary eosinophils at the level of mRNA andprotein as measured by RT-PCR and FACS. The maximum efficacy obtained onthe cell system tested, under the experimental conditions used, wasobserved at a concentration of 20 microM. In the presence of 107A, theproliferation of TF-1 cells was reduced, whether IL-3, IL-5, or GM-CSFwas used as a trophic factor. This result shows the specificity and theefficacy of 107A antisense for the beta-chain.

Eosinophil survival was inhibited by 107A in the presence of IL-5 and itappeared that apoptosis is a consequence of this inhibitory effect.Eosinophils play a key role in allergic inflammation and require GM-CSF,IL-3, and IL-5 for their differentiation, activation, and survival(Adachi et al., 1995, Am. J. Respir. Crit. Care Med. 151: 618-623 andOddera et al., 1998, Lung. 176: 237-247). In asthma, eosinophilaccumulation and survival are thought to be important contributors toinflammation and epithelial tissue damage because they release toxicproducts, including eosinophil cationic protein (Walsh et al., 1997,Clin. Exp. Allergy 27: 482-487).

Example 5 mRNA Analysis for ASM8 Target Genes in Trachea Samples

Further experiments were conducted to analyze trachea samples inCynomolgus Monkeys for the levels of mRNA for the target genes to whichASM8 is directed against (beta-chain-subunit and CCR3). On Day 15 (oneday after the last dose), trachea samples were collected immediatelyfollowing sacrifice of all Main Phase animals in Groups 1 (control) and4 (high-dose group; target dose level of 2.5 mg/kg/day) and quicklyfrozen in liquid nitrogen. The frozen trachea samples were analyzed fortarget mRNA levels by RT-PCR.

Target gene expression levels (β_(c)-subunit and CCR3) were determinedfor the monkey trachea samples using a validated, semi-quantitativeRT-PCR method. β_(c)-Subunit and CCR3-specific PCR amplifications werecarried out on trachea extracts for control and high-dose ASM8-treatedanimals (Table 7).

TABLE 7 RT-PCR Sample Analysis Results

Note: shaded values represent outliers that were not included incalculation of mean values.

Although glyceraldehyde-3-phosphate dehydrogenase (G3PDH) is typicallyused as an internal control in the analyses of RT-PCR reactions, a mildcellular infiltrate of the lungs and trachea was observed (as istypically observed with other antisense oligonucleotides at depositionsites). Thus, as the cellular infiltrate contributed to the measuredlevels of β_(c)-subunit and CCR3 (i.e., immune cells expressβ_(c)-subunit and CCR3), G3PDH was not considered to be the mostappropriate gene to use as the internal control in this case. Instead,the expression of the target genes was normalized to the mRNA levels forinflammatory cytokines; i.e., IL-4 and TNF-α. The results demonstratethat even approximately 24 hours after administration of ASM8, therelative expression of the β_(c)-subunit and CCR3 mRNA to IL-4 mRNA wasdecreased by 29% and 24%, respectively, and the expression relative toTNF-α was decreased by 30% and 24%, respectively, in ASM8-treatedanimals (Table 8).

TABLE 8 Inhibition Table Ratio β_(c)-subunit/ β_(c)-subunit/IL-4CCR3/IL-4 TNFα CCR3/TNFα Control 1.50 1.95 0.23 0.29 Treated 1.07 1.480.16 0.22 Inhibition 28.7% 24.1% 30.4% 24.1%

ASM8 treatment thus significantly inhibits the β_(c)-subunit and theCCR3 mRNA expression relative to the inflammatory cytokines IL-4 andTNF-alpha, despite the complexity of monkey tracheal tissue and the 24hours that elapsed between ASM8 dosing and obtaining the tissue samples.

Example 6 Storage Stability of ASM8

Stability testing was conducted to evaluate the integrity of theoligonucleotide constituents of ASM8 (TOP004 and TOP005) under differentstorage temperatures. This information is important to define theoptimal storage, retest, and shelf life conditions for ASM8.

Capillary gel electrophoresis (CGE) and high performance (pressure)liquid chromatography (HPLC) have been widely used for the chemicalanalysis of antisense oligonucleotides. As ASM8 consists of twooligonucleotides, the test system must provide adequate separation ofthe two individual antisense molecules. Thus, the following will bedescribed: 1) a method based on anion exchange chromatography toseparate ASM8 components (TOP 004 and TOP 005) and their degradationproducts, and 2) the effect of storage temperature on the stability ofASM8 constituents (FIGS. 14-16).

ASM8 was weighed and solubilized in PBS at a concentration of 0.5 mg/mL(0.25 mg/mL TOP004 and 0.25 mg/mL TOP 005). [The purity factor for TOP004, was 1.15 (i.e., 1.15 g of powder contains 1 g of active molecule);the purity factor for TOP 005, was 1.24 (i.e., 1.24 g of powder contains1 g of active molecule).]

To induce degradation of TOP004 and TOP005 prior to analysis (in orderto ensure resolution of degradation products from the intact molecules),the following treatments were performed:

-   -   Depurination: ASM8 was resuspended in 30% CH₃COOH at a final        concentration of 0.5 mg/mL, and incubated for 3, 4, or 6 hours        at room temperature. The reaction was stopped by addition of 5        volumes of water and the mixture placed at −20° prior to        lyophilization in a Speed-Vac to remove acetic acid.    -   Cleavage: the depurinated oligonucleotides were resuspended in        0.2 M NaOH (0.5 mg/mL), incubated at 50° C. for 1 hour, and        stored at −20° C. or analyzed by HPLC.

Aliquots of ASM8 (0.5 mg/mL) in PBS were incubated at −20° C., 4° C.,30° C., and 40° C. for 2 months. At weeks 4, and 8, the HPLC profile ofASM8 was established. The control condition was defined as the HPLCprofile of ASM8 prior to any storage time (i.e., at time zero). The HPLCsystem was driven by Breeze (V 3.30) software from Waters (FIGS. 17A1,17A2, 17B1 and 17B2).

HPLC separation was performed with a Waters 1500 Series Binary HPLC pumpcoupled to a Waters 2487 Dual λ Absorbance detector and equipped within-line degasser, oven, and 1500 series manual injector, Reodyne 7725i.

The mixture of oligonucleotides was fractionated on a Waters Protein PakDEAF 5PW anion exchange column (0.5 cm×7.5 cm), maintained at 60° C.,and detected by UV absorption at 260 nm. The oligonucleotide mixture(volume=25 microL) was loaded onto the column in water (buffer A) andthe elution was performed by progressively increasing the proportion ofbuffer B (1 M LiClO₄), resulting in an increase of ionic strength of theliquid phase (Table 9), which eluted the oligonucleotide from the solidphase (column).

Under the assay conditions, 62.5 micrograms of either TOP004 or TOP005produced a measurable change>0.15 absorbance unit (AU) at 260 nm.

TABLE 9 HPLC Gradient for Separation of ASM8 and Degradants Time (min)Flow (mL/min) Buffer A (%) Buffer B (%) 0 1 100 0 5 1 100 0 10 1 93 7100 1 65 35 102 1 20 80 122 1 20 80 124 1 100 0 144 1 100 0 146 0.1 1000

The chromatogram in FIG. 14 shows the elution profile of the individualproducts of ASM8 (TOP 004 and TOP 005) under DEAE anion exchangechromatography. A volume of 25 microL of freshly prepared ASM8 (0.5mg/mL) was fractionated on the DEAE anion exchange column. Under thegradient conditions described above, TOP004 eluted earlier than TOP 005;this is consistent with TOP004 being 2 nucleotides shorter than TOP005and having fewer negatively charged residues. The TOP004 oligonucleotideeluted at 81.3 minutes and represented 48.0% of the total materialabsorbing at 260 nm. TOP005 eluted at 86.8 minutes and represented 49.3%of the total material absorbing at 260 nm.

In order to confirm adequate separation between TOP004, TOP005, and thedegradation products of ASM8, a two-step chemical degradation of ASM8was performed. The cleavage step was kept constant but the incubationperiod for the depurination step was performed for 3 to 6 hours.Referring to FIG. 15, ASM8 (0.5 mg/mL) was treated with CH₃COOH for 3hours and submitted to alkaline lysis (as described above) prior tofractionation by DEAE anion exchange chromatography. The TOP004oligonucleotide eluted at 81.5 minutes and represented 32.4% of thetotal material absorbing at 260 nm. The TOP005 product eluted at 86.9minutes and represented 28.0% of the total material. The minor peaksrepresent degradation products of ASM8. Referring to FIG. 16, ASM8 (0.5mg/mL) was treated with CH₃COOH for 6 hours and submitted to alkalinelysis as described in above prior to fractionation by DEAE anionexchange chromatography. Under detection at 260 nm, the TOP004oligonucleotide eluted at 82.2 minutes and TOP005 eluted at 86.7minutes. By increasing the depurination time, the proportion of TOP004decreased to 20.6% and TOP005 to 14.5%. The extent of degradation ofTOP005 appeared to be slightly greater under these experimentalconditions. As seen on the chromatograms in FIGS. 15 and 16, increasingthe depurination time increased the degradation of ASM8.

Referring to FIGS. 17A1, 17A2, 17B1 and 17B2, the chemical stability ofASM8 under different storage temperatures was evaluated. ASM8 (0.5 mg/mLin PBS) was incubated at −20° C., 4° C., 30° C., or 40° C. for 4 weeks(FIGS. 17A1 and 17A2) and 8 weeks (FIGS. 17B1 and 17B2) and analyzed byDEAE anion exchange chromatography. The various storage temperaturestested in this experiment did not affect the elution profile of the ASM8components. No significant degradation of ASM8 was observed at any ofthe temperatures at which ASM8 was stored for up to 2 months.

A separation method based on DEAE anion exchange HPLC for ASM8 has beendescribed above. Because of the nature of this product, adequateseparation of the components of ASM8 (TOP004 and TOP005oligonucleotides) is preferred. Under the gradient conditions described,the retention time of TOP004 was more than 5 minutes earlier than theretention time of TOP 005, with very little overlap of the two peaks.

The method is also capable of detecting degradation products of ASM8.The chemical stability of ASM8 under different temperature, humidity,and light conditions can be assessed by this HPLC method.

The formulation of ASM8 in PBS was chemically stable, and no significantdegradation products were detected by the HPLC procedure after storageunder a range of temperatures for up to 2 months.

Example 7 Thermodynamic Evaluation of ASM8

Further experiments were conducted to ensure that the twooligonucleotide strands, TOP004 and TOP005, did not interact in solutionusing thermodynamic evaluations.

TOP004 and TOP005 were mixed at equimolar concentrations in 1×PBS (aswell as in other buffer systems). Total oligonucleotide concentrationranged from approximately 1.2 to 8.7 microM. Standard UVthermo-denaturation methods were conducted using a Beckman DU640spectrophotometer with a Tm accessory. Change in absorbance was detectedat 260 nm at each degree from 10 to 90° C. Melting curves were fittedusing MELTWIN 3.5™ software to determine thermodynamic parameters.Screen pictures of melting curves and thermodynamics summary tables wereproduced.

Referring to FIG. 18, melting curves for TOP004 and TOP005 in 1×PBS areshown. FIG. 19 is a thermodynamics summary based on results of meltingcurve fits for TOP004 and TOP005 in 1×PBS. The results demonstrated thatnone of the oligonucleotide combinations/conditions produced asignificant transition (jump in absorbance) in melting profile uponincrease in temperature. This indicated that tested oligonucleotidemixtures do not form significant secondary structure interactions attested buffer conditions.

Example 8 ASM8 Toxicity in Cynomolgus Monkey

This example shows the toxicity of ASM8, consisting of a 1:1 mixture ofTOP004 and TOP005. Also shown is the toxicokinetic profile of itsindividual oligonucleotide components, when administered by inhalationexposure once daily for 14 consecutive days to cynomolgus monkeys.Further, 14 days of inhalation exposure to ASM8 did not elicit asystemic hypersensitivity condition detectable by intradermal injection(ID).

TABLE 10 Estimated Achieved dosage Dose Group Estimated Achieved Dosage(mg/kg/day) Treatment Males Females Combined 1: Vehicle control 0 0 0 2:ASM8 Low Dose 0.05 0.05 0.05 3: ASM8 Mid Dose 0.22 0.23 0.22 4: ASM8High Dose 2.4 2.5 2.5

TABLE 11 Overall exposure aerosol concentrations Dose Group TreatmentMean (microg/L) S.D. (microg/L) C.V. (%) 1: Vehicle control 0 — — 2:ASM8 Low Dose 7.4 0.89 12.1 3: ASM8 Mid Dose 34.6 5.68 16.4 4: ASM8 HighDose 380.4 68.43 18.0

Comprehensive assessments of mortality, clinical signs, body weights,food consumption, electrocardiography, ophtalmoscopy and clinicalpathology were performed. Serial blood samples were obtained on thefirst and last days of exposure and the end of the recovery period andtissues were collected at termination, for determination of individualoligonucleotide content. Additionally, on Day 25, animals designated forthe recovery phase were given an intradermal injection (ID) of ASM8 toassess potential systemic hypersensitivity. All animals were euthanizedfollowing 14 days of exposure (Day 15) or following a 14-day recoveryperiod (Day 29) and subjected to a full necropsy with collection of acomplete set of tissues from each animal.

Histopathologic evaluation consisted of microscopic examination of alltissues from animals in the high-dose and control groups, andrespiratory tract tissues in the lower dose groups and recovery animals.

The formulation of ASM8 aerosolized readily and produced exposureaerosols that were consistently stable and respirable, with inter-groupmass median diameter (MMAD) and geometric standard deviation (GSD)values between 1.7-1.8 micrometer and 2.12-2.22, respectively. Theresultant estimated achieved doses were close to target at 0.05, 0.22and 2.5 mg/kg/day for groups 2-4, respectively, Table 7-8.

There were no deaths, and the monkeys tolerated the dosages well. Therewere no effects on body weight, food consumption, electrocardiography,ophtalmoscopy or clinical pathology parameters and hypersensitivitytesting revealed no effect of ASM8 administration. Following necropsy,organ weight measurements produced no evidence of toxicity. Macroscopicinvestigations of all organs revealed only pale discoloration to thekidneys in ASM8 treated animals. However, due to the absence ofcorroboratory microscopic alterations, clinical pathology findings ororgan weight changes, and the fact that the discoloration was not seenfollowing 14 days of recovery, this finding was considered of equivocalbiological and toxicological significance.

Plasma levels of TOP004 and TOP005, as well as their proximal (n−1)metabolites, were very low in plasma, with low- and mid dose groupsbelow the limit of quantification. For the high dose group (2.5mg/kg/day), TOP004 and TOP005 concentrations were typically greatest ateither the earliest sampling time of 0.5 hours postdose, or at the1-hour timepoint. At most postdosing timepoints, the mean concentrationof TOP004 was similar to that of TOP005, FIG. 20A and FIG. 20B.

There was no accumulation of either oligonucleotide component (or theirn−1 metabolites) in the plasma with repeated daily administration for 14days as shown in FIG. 21A and FIG. 21B There were no consistent genderdifferences in the plasma concentrations. A significant percentage ofcirculating oligonucleotide was present as the proximal n−1 metabolitefor both TOP004 and TOP005, although the percentage tended to beslightly lower for TOP004. For both oligonucleotides and their n−1metabolites, clearance from the blood compartment (plasma) was evidentover the 24-hours collection period. At the terminal sacrifice (one dayafter the last inhalation of dose of ASM8), appreciable quantities ofthe intact oligonucleotide components of ASM8 (TOP004 and TOP005) weredetected in the trachea of the high-dose animals. At the end of the14-day recovery period (Day 29), the levels of TOP004 and its n−1metabolite had diminished, relative to Day 15 and were measured slightlyabove the limit of detection of the assay. In contrast, no TOP005 or itsmetabolite were quantifiable at the recovery sacrifice timepoint. Theseresults suggest that TOP004 has greater tissue stability than TOP005.

Treatment related microscopic changes were not observed in any organexcept the respiratory tract. All the observed changes in therespiratory tract were graded on a 4 point scale as being the lowest(minimal). The changes that were noted were for the lungs included:foamy alveolar macrophages in animals dosed at 0.22 or 2.5 mg/kg/day,intra-alveolar granulocytic inflammation at 2.5 mg/kg/day, focalhemorrhage in two animals and focal bronchiolar metaplasia in one animaldosed at 2.5 mg/kg/day; for the nasal cavity: focal erosion of thesquamous epithelium of the nasal septum in 2/6 animals dosed at 2.5mg/kg/day, accompanied by acute inflammation and an inflammatory exudatein one monkey; and for the bronchial lymph nodes: foamy macrophages inanimals dosed at 2.5 mg/kg/day. The severity of the changes observed inthe lungs of mid- and high-dose animals were minor and not accompaniedby evidence of local damage or cellular infiltration in the lungparenchyma. Inflammatory cells were sparse and only seen in a smallnumber of ASM8 high-dose animals and the distribution of the changes wasconsistent with inhalation of the test material. Focal hemorrhages werevery small and interpreted as likely to be fortuitous. The changesreported are thus generally consistent with normal pulmonary mechanismsassociated with phagocytosis and clearance of an inhaled test material.Withdrawal of treatment for 14 days resulted in the continued presenceof a few foamy alveolar macrophages with no inflammation in one of thetwo ASM high-dose animals. This observation is consistent with gradualregression of the lesions and indicates that there was no progressive orpersistent alteration to the lung parenchyma.

There was no evidence of an effect of treatment on nasal tissuesfollowing the 14-day recovery period. Regarding the bronchial lymph nodefindings in high-dose animals, foamy macrophages in the medullarysinuses are consistent with clearance of the test material by lymphaticdrainage from the lung. There was no evidence of parenchymal damage, andthe lymph nodes did not appear to be in a reactive condition.

In conclusion, inhalation of ASM8 for 14 consecutive days at estimatedachieved doses of up to 2.5 mg/kg/day was well tolerated and produced noeffects on body weights, food consumption, electrocardiography, organweights, ophtalmoscopy or clinical pathology parameters, andhypersensitivity testing revealed no effect of ASM8 administration. Anumber of mostly minimal histomorphologic alterations were noted in thelungs (0.22 and 2.5 mg/kg/day), as well as in the nasal cavity andbronchial lymph nodes (2.5 mg/kg/day). These changes were reduced inseverity or absent following 14 clays of recovery.

Although preferred embodiments of the invention have been describedherein, it will be understood by those skilled in the art thatvariations may be made thereto without departing from the spirit of theinvention or the scope of the appended claims.

The invention claimed is:
 1. An antisense oligonucleotide, theoligonucleotide being directed against a nucleic acid sequence codingfor a CCR3 chemokine receptor, wherein the oligonucleotide consists ofone of (i) a sequence selected from the group consisting of SEQ ID NO. 1and SEQ ID NO. 14 and (ii) a modified oligonucleotide of a sequenceselected from the group consisting of SEQ ID NO. 1 and SEQ ID NO.
 14. 2.The antisense oligonucleotide according to claim 1, wherein theoligonucleotide consists of SEQ ID NO.
 1. 3. The antisenseoligonucleotide according to claim 1, wherein the oligonucleotideconsists of SEQ ID NO.
 14. 4. A pharmaceutical composition comprising atleast one antisense oligonucleotide as defined in claim 1, inassociation with a pharmaceutically acceptable carrier.